Co-crystal forms of a novobiocin analog and proline

ABSTRACT

Disclosed are co-crystal forms of N-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide and L-proline or D-proline, their pharmaceutical compositions, processes of manufacture, and methods of use for treating neurodegenerative disorders such as diabetic peripheral neuropathy.

CLAIM OF PRIORITY

This patent application is a continuation of U.S. patent applicationSer. No. 17/856,484, filed Jul. 1, 2022, which is a continuation of U.S.patent application Ser. No. 16/894,461, filed Jun. 5, 2020, which is acontinuation of U.S. patent application Ser. No. 16/265,256, filed Feb.1, 2019, which claims the benefit of priority to U.S. Application Ser.No. 62/627,570, filed Feb. 7, 2018, which are incorporated by referenceherein in their entireties.

BACKGROUND

Approximately 26 million Americans are afflicted with either Type 1 orType 2 diabetes. Despite the use of insulin and oral anti-diabeticmedications to help maintain euglycemia, about 60-70% of theseindividuals develop diabetic peripheral neuropathy (DPN). See Veves, A.;Backonj a, M.; Malik, R. A., Pain Med. 9 (2008) 660-674. A number ofsmall molecules based upon the novobiocin scaffold are reported toinhibit heat shock protein 90 (Hsp90), and are reported to havesignificant neuroprotective properties and to be useful for reversingsymptoms of DPN in animal models. See B. R. Kusuma et al., J. Med. Chem.55 (2012) 5797-5812; U.S. Pat. No. 9,422,320.

One novobiocin analog (“novologue”) of this type isN-(2-(5-(((3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4) which is reported to exhibit high neuronal protective activity.Kusuma (2012). Novologue 4 is further reported to have effects that aredependent upon the presence of another heat shock protein, Hsp70, whileother effects are independent of Hsp70. The precise role of Hsp70 in themechanism of action of novologue 4 and related compounds has not beenfully characterized. J. Ma et al., ACS Chem. Neurosci. 6(9) (2015)1637-1648.

The synthesis of novologue 4 is reported to follow a procedure thatresults in an amorphous solid, and its physico-chemical characterizationomits definitive assignment of stereochemistry at the 2-position (Kusuma(2012); U.S. Pat. No. 9,422,320), thereby allowing in principle for theexistence of two possible anomers 4a and 4b as shown below:

The published synthesis of 4 (also known as KU-596), while indicating anHPLC purity of 95.6%, does not indicate anomeric purity of the amorphoussolid, as evidenced by the fact that only the noviose 2-position lacksdefinitive assignment of stereochemistry. (Kusuma (2012).

SUMMARY

The present disclosure is premised upon the surprising discovery thatco-crystal forms of novologue 4a and L-proline or D-proline are realizedin high yield, purity, and anomeric purity. The inventive forms moreoverdemonstrate significant improvements in bioavailability, relative to theknown amorphous form 4.

Thus, one embodiment of the disclosure is a co-crystal ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamideand L-proline (1:2). The co-crystal is characterized by an X-ray powderdiffractogram comprising the following peaks: 14.76, 16.86, 19.00, and21.05° 2θ±0.20° 2θ as determined on a diffractometer using Cu-Kαradiation at a wavelength of 1.54178 Å. This co-crystal is referred toherein as “Form B.”

Another embodiment is a co-crystal ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamideand L-proline (1:2). The co-crystal is characterized by an X-ray powderdiffractogram comprising the following peaks: 9.20, 16.19, 18.45, and24.51° 2θ±0.2° 2θ as determined on a diffractometer using Cu-Kαradiation at a wavelength of 1.54178 Å. This co-crystal is referred toherein as “Form D.”

Additionally, an embodiment is a co-crystal ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamideand L-proline that is present as an acetone solvate (1:1:1). Thisco-crystal is characterized by an X-ray powder diffractogram comprisingthe following peaks: 14.64, 17.53, 18.91, and 21.33° 2θ±0.20° 2θ asdetermined on a diffractometer using Cu-Kα radiation at a wavelength of1.54178 Å. This co-crystal is referred to herein as “Form C.”

In a further embodiment, the present disclosure is drawn to a co-crystalofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamideand L-proline as a solvate of methyl ethyl ketone and pyrazine in amolar ratio of about 1:1.2:0.6:0.1, respectively. The co-crystal ischaracterized by an X-ray powder diffractogram comprising the followingpeaks: 10.42, 14.62, 19.28, and 21.14° 2θ±0.20° 2θ as determined on adiffractometer using Cu-Kα radiation at a wavelength of 1.54178 Å. Thisco-crystal is referred to herein as “Form G.”

The disclosure also provides a co-crystal ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamideand D-proline (1:1), characterized by an X-ray powder diffractogramcomprising the following peaks: 11.77, 14.52, 19.54, and 21.23° 2θ±0.20°2θ as determined on a diffractometer using Cu-Kai radiation at awavelength of 1.5405929 Å.

The disclosure further provides a co-crystal ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamideand L-proline (1:1), characterized by an X-ray powder diffractogramcomprising the following peaks: 8.52, 16.33, 19.50, and 21.22° 2θ±0.20°2θ as determined on a diffractometer using Cu-Kai radiation at awavelength of 1.5405929 Å.

In accordance with another embodiment, the disclosure is drawn to apharmaceutical composition that comprises any one of the co-crystalforms described herein. The composition further comprises apharmaceutically acceptable solid carrier. In some embodiments, thecomposition further comprises one or more additional co-crystal forms.

Another embodiment of the disclosure is a method for inhibiting heatshock protein 90 (Hsp90) in a subject. The method comprisesadministering to the subject a therapeutically effective amount of aco-crystal described herein.

The disclosure also is embodied in a method for treating or preventing aneurodegenerative disorder in a subject suffering therefrom. The methodcomprises administering to the subject a therapeutically effectiveamount of a co-crystal described herein. In some embodiments, theneurodegenerative disorder is diabetic peripheral neuropathy (DPN).

Alternatively, according to other embodiments, the disclosure provides amethod for preventing or reducing the likelihood of diabetic peripheralneuropathy from developing in a subject who suffers from Type 1 or Type2 diabetes. The method comprises administering to the subject atherapeutically effective amount of a co-crystal described herein.

In accordance with another embodiment, the disclosure provides a processfor making the Form B co-crystal. The process comprises the step ofheating to a first temperature a combination ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and L-proline in about a 1:1 to about a 1:2 molar ratio in aC₁₋₆-alkyl alcohol to yield a solution. The solution is then cooled to asecond temperature no higher than about 30° C. to thereby yield a slurryof the co-crystal, and the slurry is then stirred at the secondtemperature for a duration of about 72 hours or less.

Another embodiment is a process of making the Form D co-crystal. Theprocess comprises heating to a first temperature a combination ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and L-proline in about a 1:1 molar ratio in EtOH or acetonitrile,then cooling the solution to a second temperature no higher than about30° C. to thereby yield a suspension of the co-crystal. The suspensionis then stirred at the second temperature for a duration of about 72hours or less.

The disclosure is embodied in yet another process drawn to making theForm C co-crystal. The process comprises (a) optionally refluxingequimolar amounts ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and L-proline in EtOH to yield a solution, and cooling the solutionto a temperature no higher than about 30° C. to thereby yield a solidproduct. The product of step (a), or otherwise a combination ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and L-proline in about a 1:1 molar ratio, is then stirred inacetone at a temperature no higher than about 30° C. for a duration ofabout 72 hours or less to thereby yield the co-crystal.

In accordance with another embodiment, the disclosure provides a processfor making the Form G co-crystal. The process comprises combiningN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a), L-proline, and pyrazine in molar ratios of about 1:1:20,respectively, in a mixed solvent of methyl ethyl ketone (MEK) and MeOHto yield a solution, and then stirring the solution to thereby yield theco-crystal.

The disclosure additionally provides a process for making the co-crystalof 4a/D-proline as described herein, comprising heating to a firsttemperature a combination ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and D-proline in about a 1:1 molar ratio in a C₁₋₆-alkyl alcohol toyield a solution; and cooling the solution to a second temperature nohigher than about 30° C. to thereby yield a suspension of theco-crystal.

In an additional embodiment, the disclosure provides a method ofincreasing the concentration ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) relative toN-(2-(5-(((2S,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4b) in a composition comprising 4a and 4b. The method comprisescontacting the composition with proline in a solvent, and subjecting thecomposition, proline, and solvent to crystallization conditions, wherebya co-crystal of 4a and proline is produced. The bulk co-crystal exhibitsa concentration of 4a that is higher than in the composition comprising4a and 4b.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents an X-ray powder diffraction (XRPD) pattern of Form B.

FIG. 2 is a differential scanning calorimetry (DSC) curve of Form B.

FIG. 3 is a thermal gravimetric analysis (TGA) curve of Form B.

FIG. 4 is a dynamic vapor sorption (DVS) curve of Form B.

FIG. 5 is an infrared (IR) spectrum of Form B.

FIG. 6 is a Raman spectrum of Form B.

FIG. 7 is an atomic displacement ellipsoid drawing of Form B determinedby single crystal X-ray crystallography.

FIG. 8 is a calculated XRPD pattern of Form B based upon single crystalstructure determination.

FIG. 9 shows a comparison of the calculated XRPD pattern of Form B(bottom trace) to the experimental XRPD pattern of Form B (top trace).

FIG. 10 presents an X-ray powder diffraction (XRPD) pattern of Form C.

FIG. 11 is a thermal gravimetric analysis (TGA) curve of Form C.

FIG. 12 is an atomic displacement ellipsoid drawing of Form C determinedby single crystal X-ray crystallography.

FIG. 13 is a calculated XRPD pattern of Form C based upon single crystalstructure determination.

FIG. 14 presents an X-ray powder diffraction (XRPD) pattern of Form D.

FIG. 15 shows DSC (bottom trace) and TGA (top trace) curves of Form D.

FIG. 16 is a dynamic vapor sorption (DVS) curve of Form D.

FIG. 17 presents an X-ray powder diffraction (XRPD) pattern of Form G.

FIG. 18 presents an X-ray powder diffraction (XRPD) pattern of4a/D-proline co-crystal.

FIG. 19 shows DSC (bottom trace) and TGA (top trace) curves of4a/D-proline co-crystal.

FIG. 20 is a dynamic vapor sorption (DVS) curve of 4a/D-prolineco-crystal.

FIG. 21 shows mean plasma concentrations of 4a in mice followingadministration of a single oral dose of Material A (●) and amorphous 4a(◯).

FIG. 22 shows mean plasma concentrations of 4a in monkeys followingadministration of a single oral dose of Material A (●) and amorphous 4a(◯) by oral gavage, and of Material A (▾) and amorphous 4a (Δ) inloose-filled capsules.

FIG. 23 presents an X-ray powder diffraction (XRPD) pattern of4a/L-proline co-crystal Material A.

FIG. 24 shows DSC (bottom trace) and TGA (top trace) curves of4a/L-proline co-crystal Material A.

FIG. 25 is a dynamic vapor sorption (DVS) curve of 4a/L-prolineco-crystal Material A.

DETAILED DESCRIPTION Definitions

Abbreviations, acronyms, and terms as used throughout the disclosurehave the following meanings.

NMR Nuclear magnetic resonance spectroscopy OM Optical microscopy XRPDX-ray powder diffraction CP Crash precipitation FE Fast evaporation RCReaction crystallization SC Slow cooling SE Slow evaporation amt AmountAPI Active pharmaceutical ingredient B/E Birefringence and extinction eqEquivalent min. Minute(s) mol. Molar Obs Observation ppt Precipitate orprecipitation ref. Refrigerator RT Room temperature Soln/soln Solutionvac Vacuum ACN Acetonitrile 2-BuOH 2-Butanol EtOH Ethanol EtO Ac Ethylacetate IPA or 2-PrOH Isopropyl alcohol, 2-propanol IPE Isopropyl etherMEK Methyl ethyl ketone MeOH Methanol MTBE or TBME Methyl-tertiary-butylether THF Tetrahydrofuran TMP 2,3,5,6-Tetramethyl-pyrazine w/wweight/weight. The weight percentage of 4a in a 4a/proline co- crystalis calculated by excluding the proline content, i.e. w/w = (weight4a)/(weight of all non-proline species in the co-crystal).

Introduction

As summarized above, studies of novologue 4 highlighted excellentpotency of the compound in the Hsp70-independent inhibition of Hsp90(Kusuma (2012) and Ma (2015)). The studies revealed potential drawbacksto the synthesis of the compound, including its tendency to result in amixture of α-anomer 4a and β-anomer 4b and a low overall yield.Additionally, while the reported column chromatography purificationmethod of 4 is appropriate for small scale study, and even then thecompound was about 95% pure (HPLC), the method is impractical forgenerating large and pharmaceutically pure quantities of α-anomer 4a fordrug development.

The present inventors therefore undertook various crystallizationstrategies to isolate 4a. However, the inventors discovered noconditions under which 4a could be separated from 4b by crystallization.

The inventors subjected amorphous 4a to a co-crystal screen comprised of28 co-formers, and surprisingly discovered that L-proline and D-prolineselectively co-crystallized with α-anomer 4a. The inventors moreoverdiscovered that L-proline and D-proline are the only tested co-formersthat yielded any crystalline material amenable to definitivecharacterization (see Example 3).

Co-Crystal Forms

Contacting compound 4 with the co-former L-proline or D-prolinesurprisingly results in the selective co-crystallization of 4a witheither co-former (see Examples 2 and 10). In this manner,co-crystallization achieves quantities of 4 that are highly enriched in4a, relative to 4b, as determined by HPLC, for example. Thus, in anembodiment, selective co-crystallization of 4a with L-proline reducesthe concentration of the f3-anomer and it facilitates removal of minorimpurities. Consequently, formation of the 4a/L-proline co-crystalimproves the purity of 4a (HPLC) from about 90% to at least 95%, 96%,97% or 98%. Subsequent recrystallization of 4a/L-proline co-crystalfurther improves purity of 4a to at least 97%, 98%, or 99%.

Similarly, in other embodiments, co-crystallization of a startingcomposition of 4a and 4b with D-proline, such as in about equimolaramounts of 4a/4b and D-proline, results in a 4a/D-proline co-crystalwherein the purity (i.e., concentration) of 4a, relative to theconcentration of 4a in the starting composition, improves by at least15%, 10%, 5%, or 3% as determined by HPLC. Thus, for example, a startingcomposition of 4a/4b contains 4a in a concentration of about 93%, andfollowing co-crystallization with D-proline the resulting co-crystalcontains 4a in concentration of about 98%. In some embodiments, a4a/D-proline co-crystal contains 4a in a final purity of at least 85%,90%, 95%, 97%, 98%, or 99%.

In other embodiments, a quantity of the α-anomer 4a is purified bycontacting it with D-proline, such as in equimolar amounts, whereby4a/D-proline co-crystal is produced. The resultant concentration of the4a in the bulk co-crystal is higher, for instance by at least 1%, 2%,3%, 4%, or 5% (HPLC) than the concentration of 4a in the startingquantity of 4a. Each of these embodiments contemplates the optional stepof one or more re-crystallizations to even further increase the purityof 4a in a given co-crystal.

The co-crystallization also produced a variety of co-crystal forms assummarized hereinabove. The various forms are identified anddistinguished from one another by one or more analytical techniquesincluding X-ray powder diffraction (XRPD), differential scanningcalorimetry (DSC), and thermogravimetric analysis (TGA).

Form B

Thus, one embodiment denoted Form B is a co-crystal ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and L-proline in a 1:2 molar ratio, respectively. The X-ray powderdiffractogram comprises characterizing peaks at 14.76, 16.86, 19.00, and21.05° 2θ±0.2° 2θ as determined on a diffractometer using Cu-Kαradiation at a wavelength of 1.54178 Å. In an embodiment, the X-raypowder diffractogram further comprises peaks at 12.14, 17.51, 18.89, and19.41° 2θ±0.2° 2θ. In accordance with yet another embodiment, Form B isadditionally characterized substantially by its entire X-ray powderdiffractogram (see FIG. 1 ).

The DSC curve of Form B is characteristic of this co-crystal in that itexhibits an exotherm at about 211° C. According to one embodiment, FormB is characterized by the entire DSC thermogram as substantially shownin FIG. 2 .

The disclosure is further embodied in a process for making Form B (seeExample 4). The process comprises heating a combination ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and L-proline in about a 1:1 to about a 1:2 molar ratio in aC₁₋₆-alkyl alcohol to yield a solution. In some embodiments, 4a ispresent as pure 4a, while in other embodiments 4a is present incombination with the β-anomer 4b, such as the combination resulting fromthe published synthesis of 4 (Kusuma 2012, supra). For instance, 4a ispresent in 95%, 96%, 97%, 98%, or 99% (w/w). The combination is heatedto a first temperature ranging from about 50° C. to about ° C.Illustrative C₁₋₆-alkyl alcohols include methanol, ethanol, and n- andi-propanol. In one embodiment, the alcohol is ethanol. In accordancewith an embodiment, a convenient first temperature is the boiling pointof the alcohol under standard pressure. Thus, for instance, when ethanolis the alcohol, the first temperature is the boiling point, i.e., about78° C.

The process further comprises the step of cooling the solution of 4a andL-proline to a second temperature that is no higher than about 30° C. tothereby yield a slurry of the co-crystal. The slurry is stirred at thesecond temperature for a duration of about 72 hours or less. In anembodiment, the slurry is filtered to isolate Form B.

Form D

The disclosure is further embodied in a co-crystal of 4a and L-prolinepresent in a 1:2 molar ratio, respectively, and it is denoted as Form D.Form D is characterized by an X-ray powder diffractogram comprising thefollowing peaks: 9.20, 16.19, 18.45, and 24.51° 2θ±0.2° 2θ as determinedon a diffractometer using Cu-Kα radiation at a wavelength of 1.54178 Å.A further embodiment is drawn to additional characterizing peaksoccurring at 11.83, 17.16, 20.15, and 25.34° 2θ±0.2° 2θ. Form D isadditionally characterized by its X-ray powder diffractogram assubstantially shown in FIG. 14 .

The DSC curve of Form D also is characteristic of this co-crystal inthat it exhibits an endotherm at about 212.2° C., with an onsettemperature of about 211.2° C. According to an embodiment, Form D ischaracterized by the entire DSC thermogram as substantially shown inFIG. 15 .

An embodiment of the disclosure also relates to a process for makingForm D. The process comprises the step of heating to a first temperaturea combination ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and L-proline in about a 1:1 molar ratio in EtOH or acetonitrile.In some embodiments, 4a is present as pure 4a, while in otherembodiments 4a is present in combination with the β-anomer 4b, such asthe combination resulting from the published synthesis of 4 (Kusuma2012, supra). For instance, 4a is present in 95%, 96%, 97%, 98%, or 99%(w/w). The first temperature is one selected in the range of about ° C.to about 85° C. A convenient temperature, for example, is achieved byrefluxing the combination, i.e., at the boiling point of acetonitrile ofabout 82° C.

The process further comprises the steps of cooling the solution to asecond temperature no higher than about 30° C. to thereby yield asuspension of the co-crystal, and then stirring the suspension at thesecond temperature for a duration of about 72 hours or less. Accordingto an embodiment, the suspension is filtered, for example, to isolateForm D.

Form C

The disclosure is further embodied in an acetone solvate of a co-crystalof 4a and L-proline present in 1:1:1 molar ratios, respectively, and itis denoted as Form C (see Example 6). The co-crystal is characterized byan X-ray powder diffractogram comprising the following peaks: 14.64,17.53, 18.91, and 21.33° 2θ±0.2° 2θ as determined on a diffractometerusing Cu-Kα radiation at a wavelength of 1.54178 Å. More specifically,in accordance with another embodiment, the X-ray powder diffractogramcomprises additional peaks at 12.10, 15.14, 18.26, and 19.56° 2θ±0.2°2θ. These and additional peaks that are characteristic of Form C areexhibited in its X-ray powder diffractogram as substantially shown inFIG. 10 .

Form C is additionally characterized by reference to its TGA thermogramthat comprises weight loss steps concluding at about 150° C. and about220° C. An embodiment is drawn to the TGA thermogram of Form C, assubstantially shown in FIG. 11 .

Form C is made by a process in accordance with various embodiments ofthe disclosure. Thus, in one embodiment, the process comprises refluxingequimolar amounts ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and L-proline in EtOH to yield a solution, and then cooling thesolution to a temperature no higher than about 30° C. to thereby yield asolid product. The solid product is then stirred in acetone at atemperature no higher than about 30° C. for a duration of about 72 hoursor less to thereby yield Form C.

Alternatively, a combination of 4a and L-proline in about a 1:1 molarratio is stirred in acetone at a temperature no higher than about 30° C.for a duration of about 72 hours or less to thereby yield Form C. Ineither of these embodiments, 4a is present as pure 4a or as acombination with the β-anomer 4b, such as that produced by the publishedsynthesis of 4. In a further embodiment, Form C is isolated, such as byfiltration.

Form G

The disclosure further relates to a co-crystal of 4a and L-proline thatexists as a solvate of methyl ethyl ketone and pyrazine and it isdenoted as Form G (see Example 9). As explained in the examples, XRPDindexing of Form G is consistent with a 4a:L-proline molar ratio of 1:1,but the indexing does not distinguish between the comparably sized MEKand pyrazine molecules, making definitive amounts of the solventsdifficult to establish by this analytical technique. Proton NMR analysisof Form G, however, established molar ratios of 4a, L-proline, MEK, andpyrazine at about 1:1.2:0.6:0.1, respectively. Form G is thuscharacterized by its XRPD diffractogram having characterizing peaks at10.42, 14.62, 19.28, and 21.14° 2θ±0.2° 2θ as determined on adiffractometer using Cu-Kα radiation at a wavelength of 1.54178 Å. Afurther embodiment provides additional peaks at 11.85, 14.93, 17.40, and19.28° 2θ±0.2° 2θ. Form G can also be characterized by its full XRPDdiffractogram as substantially shown in FIG. 17 .

XRPD analysis of Form G further established unit cell parameters thatcharacterize the co-crystal, according to another embodiment. Thus, theparameters are a=10.975 Å, b=10.310 Å, c=15.704 Å, α=90°, β=108.56°, andγ=90°.

The disclosure further relates to a process for making Form G. Theprocess comprises combiningN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a), L-proline, and pyrazine in molar ratios of about 1:1:20,respectively, in a mixed solvent of methyl ethyl ketone (MEK) and MeOHto yield a solution. In some embodiments, 4a is present as pure 4a,while in other embodiments 4a is present in combination with theβ-anomer 4b, such as the combination resulting from the publishedsynthesis of 4 (Kusuma 2012, supra). For instance, 4a is present in 95%,96%, 97%, 98%, or 99% (w/w). In general, the mixed solvent is embodiedby an excess of MEK over MeOH. Thus, an illustrative ratio of MEK toMeOH is about 9:1 (v/v). The solution is then stirred to thereby yieldForm G.

Material A

Another embodiment of the present disclosure is a co-crystal of 4a andL-proline present in a 1:1 molar ratio, respectively, and it is denotedas Material A. Material A is characterized by an X-ray powderdiffractogram comprising the following peaks: 8.52, 16.33, 19.50, and21.22° 2θ±0.20° 2θ as determined on a diffractometer using Cu-Kairadiation at a wavelength of 1.5405929 Å. A further embodiment is drawnto additional characterizing peaks occurring at 9.19, 13.22, 14.75, and17.57° 2θ±0.2° 2θ. Material A is additionally characterized by its X-raypowder diffractogram as substantially shown in FIG. 23 .

XRPD analysis of Material A further established unit cell parametersthat characterize the co-crystal, according to another embodiment. Thus,the parameters are a=10.126 Å, b=11.021 Å, c=30.259 Å, α=90°, β=90°, andγ=90°.

The DSC curve of Material A also is characteristic of this co-crystal inthat it exhibits an endotherm at about 145° C. According to anembodiment, Material A is characterized by the entire DSC thermogram assubstantially shown in FIG. 24 .

Material A is additionally characterized by reference to its TGAthermogram that comprises weight loss steps concluding at about 160° C.and about 230° C. An embodiment is drawn to the TGA thermogram ofMaterial A, as substantially shown in FIG. 24 .

4a/D-Proline Co-Crystal

The disclosure also provides in another embodiment a co-crystal of 4aand D-proline present in a 1:1 molar ratio (see Example 11). Theco-crystal is characterized by its XRPD diffractogram havingcharacterizing peaks at 11.77, 14.52, 19.54, and 21.23° 2θ±0.20° 2θ asdetermined on a diffractometer using Cu-Kai radiation at a wavelength of1.5405929 Å. Additional characterizing peaks occur at 8.45, 13.18,16.95, and 19.12° 2θ±0.2° 2θ. These and even additional peaks that arecharacteristic of the co-crystal are exhibited in its X-ray powderdiffractogram as substantially shown in FIG. 18 .

The DSC curve of the 4a/D-proline crystal also is characteristic of thisco-crystal in that it exhibits an endotherm at about 130° C. Accordingto an embodiment, the co-crystal is characterized by the entire DSCthermogram as substantially shown in FIG. 19 .

The co-crystal is additionally characterized by reference to its TGAthermogram that comprises two weight loss steps concluding at about150-160° C. and about 230° C., respectively. An embodiment is drawn tothe TGA thermogram of the co-crystal, as substantially shown in FIG. 19.

The disclosure further provides a process for making the 4a/D-prolinecrystal. The process comprises the steps of (a) heating to a firsttemperature a combination ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamide(4a) and D-proline in about a 1:1 molar ratio in a C₁₋₆-alkyl alcohol toyield a solution; and (b) cooling the solution to a second temperatureno higher than about 30° C. to thereby yield a suspension of theco-crystal. In some embodiments, 4a is present as pure 4a, while inother embodiments 4a is present in combination with the 13-anomer 4b,such as the combination resulting from the published synthesis of 4(Kusuma 2012, supra). For instance, 4a is present in 95%, 96%, 97%, 98%,or 99% (w/w).

Methods of Purifying

The surprising discovery that 4a selectively co-crystallizes with L- andD-proline engenders, according to an embodiment, a method for purifying4a from bulk quantities of 4. That is to say, the co-crystallization of4a with proline enriches the concentration of 4a relative to 4b in aresulting bulk sample of a 4a/proline co-crystal. A method to increasethe concentration of 4a first contemplates a starting composition of 4aand 4b. The starting composition can be the bulk solid that results fromthe published synthesis of 4, or by one of a number of alternativesynthetic pathways, known or reasonably contemplated by those skilled inthe art of organic synthesis, that lead to 4. Additionally, the startingcomposition can be a bulk solid of predominantly 4a that resulted fromother means of purification, such as column chromatography. Theinventors surprisingly discovered in this regard that 4a defied allattempts at crystallization; in fact, under no conditions was 4aobserved to exist in crystalline form. In any of these examples, thestarting composition contains at least some amount of 4b, such as 0.5 toabout 10% (w/w).

A molar excess of proline, such as one to about two equivalents, iscombined with the starting composition in a solvent. In someembodiments, the proline is L-proline, and in other embodiments theproline is D-proline. It is possible to use mixtures of L- andD-proline. Any solvent capable of substantially dissolving proline andstarting composition is suitable for this purpose. Exemplary solvents,such as any solvent described herein, include C₁-C₆-alkyl alcohols, suchas methanol and ethanol. In some embodiments of the method, it isadvantageous to promote dissolution by heating the starting composition,proline, and solvent mixture. A convenient temperature for this purposeis the reflux temperature of the solvent.

The combination of starting composition, proline, and solvent is thensubjected to crystallization conditions to achieve co-crystallization of4a and proline. Various crystallization techniques are useful in thiscontext, such as any of those described herein. In exemplaryembodiments, a warm solution of the starting composition and proline isallowed to cool to room temperature. External cooling measures can beimplemented to cool the solution below room temperature to facilitateco-crystallization. Alternatively, or in combination, the solvent isallowed to slowly evaporate. Any of these means, alone or in combinationwith each other, disturb solution equilibrium toward crystallization.

The resulting bulk co-crystal of 4a and proline is thereby enriched in4a, relative to the concentration of 4a in the starting composition. Thecorresponding concentration of 4b is decreased. In addition, the methodpurifies 4a from other impurities. A convenient method for quantifyingthe concentration of 4a is by HPLC, although any analytical techniquecapable of resolving and quantifying the components present in themixture would be suitable for this purpose, including gas chromatography(GC) conducted on chiral stationary phases. Thus, for instance, theconcentration of 4a in the bulk co-crystal is about 3 to about 20%, orabout 5 to about 15% (w/w) higher than in the starting composition.Alternatively, the increase in 4a in the bulk co-crystal, relative tothe concentration of 4a in the bulk starting composition, is at leastabout 5%, about 10%, or about 15% (w/w). Thus, for example, a startingcomposition of 4 contains about 93% 4a and about 6% 4b, as determined byHPLC (see Example 10(A)). Following co-crystallization with L-proline asprescribed by the inventive method, the amount of 4b in the resultingbulk co-crystal decreases to about 2.5%. In any of these embodiments,subsequent recrystallization of the 4a/proline co-crystal can furtherdecrease the amount of 4b in the bulk material.

This disclosure refers to patterns, such as XRPD patterns, in terms oftheir characteristic peaks. The assemblage of such peaks is unique to agiven co-crystal form within the uncertainties attributable toindividual instruments and to experimental conditions. Thus, forinstance, each XRPD peak is disclosed in terms of an angle 2θ that hasan acceptable uncertainty of ±0.2° 2θ, it being therefore understoodthat variances of characteristic peaks within this uncertainty in no wayundercut the identity of a co-crystal form with a correspondingassemblage of its characteristic peaks.

Pharmaceutical Composition

The disclosure also contemplates as another embodiment a pharmaceuticalcomposition that comprises a co-crystal as described herein. Asexplained in the examples, the inventive co-crystal surprisinglyexhibits much greater bioavailability than 4a alone, i.e., as theamorphous solid. Therefore, the pharmaceutical composition can beformulated to contain a lower concentration of co-crystal to achievetherapeutically the same effects, relative to formulations that containamorphous 4a. By this benefit of the inventive co-crystal,therapeutically effective amounts of a co-crystal in a pharmaceuticalcomposition provide a dose of about 0.1 mg to about 1000 mg, adjusted asnecessary according to the weight of a subject. Typical dosages can varyfrom about 0.01 mg/kg to about 100 mg/kg per day.

The pharmaceutical composition further comprises, in accordance withaccepted practices of pharmaceutical compounding, one or morepharmaceutically acceptable excipients, diluents, adjuvants,stabilizers, emulsifiers, preservatives, colorants, buffers, or flavorimparting agents, that in aggregate constitute a pharmaceuticallyacceptable carrier. In general, the pharmaceutical composition isprepared with conventional materials and techniques, such as mixing,blending, and the like. In principle, the pharmaceutically acceptablecarrier can be a liquid so long as the co-crystal maintains constitutiveand structural stability, such as by not dissolving in the carrier. Ingeneral, however, the pharmaceutically acceptable carrier and, hence,the composition as a whole are solids.

In accordance with some embodiments, the pharmaceutical compositionfurther comprises one or more additional co-crystals as disclosedherein. For example, the composition comprises two forms, three forms,or four forms. An exemplary composition comprises Form B and Form D.Binary compositions, i.e., those containing just two forms, provide theforms in various weight ratios ranging from about 0.05:1 to about1:0.05. Intermediate ratios and ranges also are contemplated, such as0.2:1 to about 1:0.2, and 0.5:1 to about 1:0.5.

For tablet compositions, an inventive co-crystal in admixture withnon-toxic pharmaceutically acceptable excipients is used for themanufacture of tablets. Examples of such excipients include withoutlimitation inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginic acid;binding agents, for example starch, gelatin or acacia, and lubricatingagents, for example magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known coatingtechniques to delay disintegration and absorption in thegastrointestinal tract and thereby to provide a sustained therapeuticaction over a desired time period. For example, a time delay materialsuch as glyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil. Theseformulations, and all other liquid formulations described herein, aresubject to the limitations delineated above for preserving constitutiveand structural integrity of the solid co-crystal.

The pharmaceutical composition is presented as a suspension inaccordance with embodiments described below. The embodiments refer to a“stable suspension,” meaning that a given co-crystal or combination ofco-crystals maintains its characteristic features, e.g., XRPD peaks,even while in contact with other components of the suspension, i.e., bynot dissolving in the liquid excipients of a suspension, not convertingto another co-crystal or amorphous form, or both.

For aqueous suspensions the inventive co-crystal is admixed withexcipients suitable for maintaining a stable suspension. Examples ofsuch excipients include, without limitation, sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.

Oral suspensions can also contain dispersing or wetting agents, such asnaturally-occurring phosphatide, for example, lecithin, or condensationproducts of an alkylene oxide with fatty acids, for examplepolyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for exampleethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin, or cetyl alcohol.

Sweetening agents such as those set forth above, and flavoring agentsmay be added to provide palatable oral preparations. These compositionsmay be preserved by the addition of an anti-oxidant such as ascorbicacid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water can provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanthnaturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitan monooleate. The emulsions may also contain sweetening andflavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, and flavoring and coloringagents. The pharmaceutical compositions may be in the form of a sterileinjectable, an aqueous suspension or an oleaginous suspension. Thissuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation may also besterile injectable solution or suspension in a non-toxic, parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The inventive co-crystals may also be administered in the form ofsuppositories for rectal administration of the co-crystal. Thesecompositions can be prepared by mixing the co-crystal with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the co-crystal. Examples of such materials are cocoa butterand polyethylene glycols.

Compositions for parenteral administrations are administered in asterile medium. Depending on the vehicle used and the concentration ofthe co-crystal in the formulation, the parenteral formulation can be asuspension of the co-crystal provided that particle size distribution ofthe co-crystal is appropriate for this mode of administration. Adjuvantssuch as local anesthetics, preservatives and buffering agents can alsobe added to parenteral compositions.

Methods of Use

One surprising advantage conferred by the inventive co-crystals, asevidenced by the appended examples, is the ability to manufacture largequantities of 4a in very high diastereomeric and chemical purities. Thisis especially important for the development of 4a in compliance, forexample, with Good Manufacturing Practice (GMP) regulations promulgatedby the U.S. Food and Drug Administration. By contrast, synthesis ofamorphous 4a requires subsequent and laborious separation techniques,such as chromatography—and all crystallization attempts wereunsuccessful as mentioned above—that are still inefficient at isolating4a in high chemical and diastereomeric purities for GMP purposes. Forthese reasons, the inventive co-crystals and processes for making themprovide large quantities of 4a that are useful in clinical trials andcommercialization efforts.

Another advantage of the inventive co-crystals resides in theunexpectedly high bioavailability of 4a from the co-crystals incomparison to amorphous 4a. More specifically, in vivo administration ofa co-crystal increased the bioavailability of 4a by a factor of about1.5-2, relative to the same dose of amorphous 4a (see Examples 12 and13). This feature of the inventive co-crystals is all the moresurprising in view of, and in fact it stands in contrast to, the generalobservation that amorphous forms of pharmaceuticals are markedly moresoluble and, hence, more bioavailable, than their crystallinecounterparts. See, B. C. Hancock et al., Pharm. Res. 17(4) (2000)397-404; B. C. Hancock et al., J. Pharm. Sci. 86(1) (1997) 1-12.

In light of these advantages, the present disclosure is further drawn tothe use of any of the co-crystal forms, including pharmaceuticalcompositions thereof, for treating or preventing a neurodegenerativedisorder in a subject that suffers from the disorder. Non-limitingexamples of such neurodegenerative disorders include Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS),Huntington's disease, spinal muscular atrophy, spinocerebellar ataxiaand forms of ataxia, and demyelinating nerve disorders including motorneuron diseases. In addition, the co-crystal forms and theirpharmaceutical compositions are useful in the treatment of diabeticneuropathy (including both the painful and insensate forms thereof) andother forms of neuropathy, including neuropathic pain that is not due todiabetes.

The co-crystal forms and their pharmaceutical compositions are alsouseful in treating neurological disorders involving mitochondrialdysfunction, oxidative stress, or inflammation, in light of the reporteduse of compound 4a to improve impaired mitochondrial function in neuronsand to reduce the expression of inflammatory markers in diabetic neurons(Ma (2015)). Because diabetic tissues undergo significant oxidativestress, these results indicate that the inventive co-crystal forms andtheir pharmaceutical compositions are useful in treating otherneurological disorders that involve oxidative stress and chronicinflammation, including epilepsy, multiple sclerosis, spinal cordinjury, and psychiatric disorders including schizophrenia, depression,bipolar disorder, autism and related disorders, and post-traumaticstress disorders. The compositions and co-crystal forms can be used incombination with other therapies, particularly therapies that reduceoxidative stress, inflammation, and mitochondrial dysfunction by othermechanisms.

As the term is used herein, “neurodegenerative disorder” refers to adisorder in which progressive loss of neurons occurs either in theperipheral nervous system or in the central nervous system. Thus, in oneembodiment, the disclosure provides a method for inhibiting Hsp90 in asubject, such as during or pursuant to the treatment of theneurodegenerative disorder, by inhibiting the progressive deteriorationof neurons that leads to cell death.

A method as described herein comprises administering to the subject atherapeutically effective amount of an inventive co-crystal. Within thedosing guidelines set forth above, a “therapeutically effective amount”is an amount of a co-crystal that inhibits, totally or partially, theprogression of the disorder or alleviates, at least partially, one ormore symptoms of the disorder. A therapeutically effective amount canalso be an amount that is prophylactically effective. The amount that istherapeutically effective will depend upon the patient's size andgender, the disorder to be treated, the severity of the disorder and theresult sought. For a given patient and disorder, a therapeuticallyeffective amount can be determined by methods known to those of skill inthe art.

In various embodiments, a method entails prevention of a neurologicaldisorder. The term “preventing” or “prevention” as used herein meansthat an inventive co-crystal is useful when administered to a subjectwho has not been diagnosed as possibly having the disorder at the timeof administration, but who would normally be expected to develop thedisorder or be at increased risk for the disorder. An inventiveco-crystal slows the development of the disorder symptoms, delays theonset of the disorder, or prevents the subject from developing thedisorder at all. Prevention also contemplates the administration of aco-crystal to a subject who is thought to be predisposed to the disorderdue to age, familial history, genetic or chromosomal abnormalities,and/or due to the presence of one or more biological markers for thedisorder.

In other embodiments, an inventive method entails “treatment” or“treating,” meaning that a co-crystal is used in a subject with at leasta tentative diagnosis of the disorder. Hence, the co-crystal of theinvention delays or slows the progression of the disorder. In addition,the term “treatment” embraces at least an amelioration of the symptomsassociated with the disorder, where amelioration is used in a broadsense to refer to at least a reduction in the magnitude of a parameter,e.g. symptom, associated with the condition being treated. As such,“treatment” also includes situations where the disorder, or at leastsymptoms associated therewith, are completely inhibited, e.g. preventedfrom happening, or stopped, e.g. terminated, such that the subject nolonger suffers from the disorder, or at least the symptoms thatcharacterize the disorder.

In one embodiment, the neurodegenerative disorder is sensory neuronglucotoxicity resultant from, e.g., hyperglycemia associated with adiabetic condition. For example, a subject suffers from Type 1 or Type 2diabetes. More specifically, in accordance with an embodiment, theneurodegenerative disorder is diabetic peripheral neuropathy. Thus, inan embodiment, an inventive method comprises preventing or reducing thelikelihood of diabetic peripheral neuropathy from developing in asubject who suffers from Type 1 or Type 2 diabetes.

In the context of the inventive methods and uses, the “subject” to betreated with an inventive co-crystal is an animal and is preferably amammal, e.g., dogs, cats, mice, monkeys, rats, rabbits, horses, cows,guinea pigs, sheep. In an embodiment, the subject is a human.

EXAMPLES

The following non-limiting examples are provided to illustrateadditional embodiments of the present disclosure.

Amorphous 4a in about 95% purity (HPLC) is obtained, for example, inaccordance with published procedures (Kusuma (2012) and U.S. Pat. No.9,422,320).

I. General Crystallization Experimental Methods

Crash Precipitation (CP): Solutions of 4a were prepared in varioussolvents or solvent systems with various coformers in given molar ratioswith agitation. Aliquots of various antisolvents were dispensed withstirring until precipitation occurred. Mixtures were allowed to stir fora specified period of time. Where stated, additional crystallizationtechniques were employed.

Fast Evaporation (FE): Solutions of 4a were prepared in various solventswith various coformers in given molar ratios with agitation. Eachsolution was allowed to evaporate from an open vial at ambientconditions unless otherwise stated. Solutions were allowed to evaporateto dryness unless designated as partial evaporations (solid present witha small amount of solvent remaining), in which case solids were isolatedby the stated method or additional crystallization techniques wereemployed as stated.

Manual Grinding: Weighed amounts of 4a and various coformers weretransferred to an agate mortar. A small amount of a given solvent wasadded to the solids, and the mixtures were manually ground with an agatepestle for a given amount of time.

Milling: Weighed amounts of 4a and given coformers were transferred toagate milling containers. A small amount of given solvent and an agatemilling ball were added to the containers, which were then attached to aRetsch mill. The mixtures were milled at 30 s⁻¹ for the stated duration.The solids were scraped down the walls of the jar between cycles.

Reaction Crystallization (RC): Mixtures of 4a with various coformerswere prepared in a given solvent by adding solids of one component to asolution of the second component. When enough solids were added suchthat the solution contained differing concentrations of each component(generally a 10- to 20-fold difference in molarity of one component vs.the other), the solution was allowed to stir for an extended period oftime. When specified, additional solids of the more concentratedcomponent were added if no precipitation occurred, and the mixture wasagain allowed to stir for an extended period of time. Any precipitatedsolids were isolated and analyzed.

Slow Cool (SC): Concentrated solutions of 4a were prepared in varioussolvent systems with various coformers in given molar ratios at elevatedtemperatures with stirring. Each vial was capped and left on the hotplate, and the hot plate was turned off to allow the sample to slowlycool to ambient temperature. If no solids were present after cooling toambient temperature, the sample was placed in the refrigerator(approximately 2 to 8° C.) and/or the freezer (approximately −10 to −25°C.) for further cooling. If no solids were present, additionalcrystallization techniques were employed, as specified.

Slow Evaporation (SE): Solutions of 4a were prepared in various solventsystems with various coformers in given molar ratios. Each solution wasallowed to evaporate at ambient conditions in a vial covered withaluminum foil perforated with pinholes. Solutions were allowed toevaporate to dryness unless designated as partial slow evaporations, inwhich a portion of the solvent evaporated. Resulting solids wereisolated by the stated technique or additional crystallizationtechniques were employed, where stated.

Slurry Experiments: Suspensions of 4a with various coformers in statedmolar ratios were prepared by adding enough solids to a given solventsystem at ambient conditions such that undissolved solids were present.The mixtures were then agitated (typically by stirring) in a sealed vialat the stated conditions for an extended period of time. Solids werecollected by the stated technique or additional crystallizationtechniques were employed where stated.

Vapor Diffusion (VD): Concentrated solutions of given starting materials(either a given form of 4a/L-proline or stated stoichiometric mixturesof 4a and L-proline) were prepared in various solvents. In some cases,solutions were filtered through a 0.2-μm nylon filter. Each solution wasdispensed into a small vial, which was then placed inside a larger vialcontaining a given antisolvent. Where stated, seeds of a given4a/L-proline form were added to the solutions. The small vial was leftuncapped and the larger vial was capped to allow vapor diffusion tooccur. Where stated, additional crystallization techniques wereattempted.

Vacuum Filtration: Solids were collected on paper or nylon filters byvacuum filtration and air dried on the filters under reduced pressurebriefly before transferring to a vial.

Interconversion Slurries: Solutions of given starting materials (eithera given 4a/L-proline form or stated stoichiometric mixtures of 4a andL-proline) were prepared by adding solids to a given solvent system at astated temperature. If a saturated solution was specified, thesuspension was agitated at ambient temperature for an extended period oftime to ensure saturation of the liquid phase. Seed crystals of each ofthe given 4a/L-proline forms of interest were added to the preparedsolutions (or to the filtered liquid phase from a saturated solution)such that undissolved solids were present. Each mixture was thenagitated (typically by stirring) in a sealed vial at a statedtemperature for a given duration. The solids were isolated by vacuumfiltration and analyzed.

II. X-Ray Powder Diffraction (XRPD) Peak Identification

Throughout this disclosure are x-ray diffraction patterns and tableswith peak lists. Peaks within the range of up to about 30° 2θ wereselected. Rounding algorithms were used to round each peak to thenearest 0.01° 2θ. The location of the peaks along the x-axis (° 2θ) inboth the figures and the lists were determined using proprietarysoftware (TRIADS™ v2.0) and rounded to two significant figures after thedecimal point. Peak position variabilities are given to within ±0.2° 2θbased upon recommendations outlined in the USP discussion of variabilityin x-ray powder diffraction (United States Pharmacopeia, USP 38-NF 33through S2, <941>Dec. 1, 2015). The accuracy and precision associatedwith any particular measurement disclosed herein has not beendetermined. Moreover, third party measurements on independently preparedsamples on different instruments may lead to variability which isgreater than ±0.2° 2θ. The wavelength used to calculate d-spacings was1.5405929 Å, the Cu-K_(α1) wavelength (Holzer, G.; Fritsch, M.; Deutsch,M.; Hartwig, J.; Forster, E. Phys. Rev. 1997, A56 (6), 4554-4568).Variability associated with d-spacing estimates was calculated from theUSP recommendation, at each d-spacing, and provided in the respectivedata tables.

Per USP guidelines, variable hydrates and solvates may display peakvariances greater than 0.2° 2θ and therefore peak variances of 0.2° 2θare not applicable to these materials.

For samples with only one XRPD pattern and no other means to evaluatewhether the sample provides a good approximation of the powder average,peak tables contain data identified only as “Prominent Peaks”. Thesepeaks are a subset of the entire observed peak list. Prominent peaks areselected from observed peaks by identifying preferably non-overlapping,low-angle peaks, with strong intensity.

Where multiple diffraction patterns are available, then assessments ofparticle statistics (PS) and/or preferred orientation (PO) are possible.Reproducibility among XRPD patterns from multiple samples analyzed on asingle diffractometer indicates that the particle statistics areadequate. Consistency of relative intensity among XRPD patterns frommultiple diffractometers indicates good orientation statistics.Alternatively, the observed XRPD pattern may be compared with acalculated XRPD pattern based upon a single crystal structure, ifavailable. Two-dimensional scattering patterns using area detectors canalso be used to evaluate PS/PO. If the effects of both PS and PO aredetermined to be negligible, then the XRPD pattern is representative ofthe powder average intensity for the sample and prominent peaks may beidentified as “Representative Peaks”. In general, the more datacollected to determine Representative Peaks, the more confident one canbe of the classification of those peaks.

“Characteristic peaks”, to the extent they exist, are a subset ofRepresentative Peaks and are used to differentiate one crystallinepolymorph from another crystalline polymorph (polymorphs beingcrystalline forms having the same chemical composition). Characteristicpeaks are determined by evaluating which representative peaks, if any,are present in one crystalline polymorph of a compound against all otherknown crystalline polymorphs of that compound to within ±0.2° 2θ. Notall crystalline polymorphs of a compound necessarily have at least onecharacteristic peak.

Instrumental Techniques

Differential Scanning calorimetry (DSC): DSC was performed using a TAInstruments Q2000 differential scanning calorimeter. Temperaturecalibration was performed using NIST-traceable indium metal. A samplewas placed into an aluminum Tzero crimped DSC pan, covered with a lid,and the weight was accurately recorded. A weighed aluminum panconfigured as the sample pan was placed on the reference side of thecell.

Dynamic Vapor Sorption (DVS): Dynamic vapor sorption data were collectedon a VTI SGA-100 Vapor Sorption Analyzer. NaCl and polyvinypyrrolidone(PVP) were used as calibration standards. Samples were not dried priorto analysis. Sorption and desorption data were collected over a rangefrom 5% to 95% RH at 10% RH increments under a nitrogen purge. Theequilibrium criterion used for analysis was less than 0.0100% weightchange in 5 minutes with a maximum equilibration time of 3 hours. Datawere not corrected for the initial moisture content of the samples.

EasyMax™ Reactor: Crystallization experiments were performed using theMettler Toledo EasyMax™ 102 with Julabo F26 chiller/circulator.Crystallizations were performed in 20 mL glass tubes (capped) withmagnetic stirring. The temperature was controlled using the jackettemperature (Tj) setting.

Elemental Analysis: Elemental analyses were carried out by GalbraithLaboratories, Knoxville, TN.

Infrared Spectroscopy: IR spectra were acquired on Nicolet 6700 Fouriertransform infrared (FT-IR) spectrophotometer (Thermo Nicolet) equippedwith an Ever-Glo mid/far IR source, a potassium bromide (KBr)beamsplitter, and a deuterated triglycine sulfate (DTGS) detector.Wavelength verification was performed using NIST SRM 1921b(polystyrene). An attenuated total reflectance (ATR) accessory(Thunderdome™, Thermo Spectra-Tech), with a germanium (Ge) crystal wasused for data acquisition. Each spectrum represents 256 co-added scanscollected at a spectral resolution of 4 cm⁻¹. A background data set wasacquired with a clean Ge crystal. A Log 1/R (R=reflectance) spectrum wasobtained by taking a ratio of these two data sets against each other.

Raman Spectroscopy: Raman spectra were acquired on a FT-Raman moduleinterfaced to a Nexus 670 FT-IR spectrophotometer (Thermo Nicolet)equipped with an indium gallium arsenide (InGaAs) detector. Wavelengthverification was performed using sulfur and cyclohexane. Each sample wasprepared for analysis by placing the sample into a pellet holder.Approximately 0.514 W of Nd:YVO₄ laser power (1064 nm excitationwavelength) was used to irradiate the sample. Each spectrum represents256 co-added scans collected at a spectral resolution of 4 cm⁻¹.

Single Crystal X-Ray Diffraction (SCXRD): The single crystal structuresof 4a/L-proline Form B and Form C were determined at the CrystallographyLaboratory at Purdue University.

Thermogravimetry (TGA): TG analyses were performed using a TAInstruments 2050 or a Discovery thermogravimetric analyzer. Temperaturecalibration was performed using nickel and Alumel™. Each sample wasplaced in an aluminum or platinum pan and inserted into the TG furnace.The furnace was heated under a nitrogen purge from ambient temperatureto 350° C. at a heating rate of 10° C./min.

Optical Microscopy: Samples were observed under a Wolfe opticalmicroscope with crossed polarizers at either 2× or 4× objectives orunder a Leica stereomicroscope with a first order red compensator withcrossed polarizers at 0.8× to 10× objectives.

Solution ¹H NMR Spectroscopy: The solution ¹H NMR spectra were acquiredby Spectral Data Services of Champaign, IL at 25° C. with a Varian^(UNITY)INOVA-400 spectrometer. The samples were dissolved in DMSO-d6.The residual peak from incompletely deuterated DMSO is at approximately2.5 ppm, and a relatively broad peak at approximately 3.3 ppm is due towater.

X-Ray Powder Diffraction (XRPD)

PANalytical X′PERT Pro MPD Diffractometer—Transmission Geometry (MostSamples): XRPD patterns were collected with a PANalytical X′Pert PRO MPDdiffractometer using an incident beam of Cu radiation produced using anOptix long, fine-focus source. An elliptically graded multilayer mirrorwas used to focus Cu Kα X-rays through the specimen and onto thedetector. Prior to the analysis, a silicon specimen (NIST SRM 640e) wasanalyzed to verify the observed position of the Si 111 peak isconsistent with the NIST-certified position. A specimen of the samplewas sandwiched between 3-μm-thick films and analyzed in transmissiongeometry. A beam-stop, short antiscatter extension, and antiscatterknife edge were used to minimize the background generated by air. Sollerslits for the incident and diffracted beams were used to minimizebroadening from axial divergence. Diffraction patterns were collectedusing a scanning position-sensitive detector (X′Celerator) located 240mm from the specimen and Data Collector software v. 2.2b.

PANalytical X′PERT Pro MPD Diffractometer—Reflection Geometry (Samplesin Limited Quantity): XRPD patterns were collected with a PANalyticalX′Pert PRO MPD diffractometer using an incident beam of Cu Kα radiationproduced using a long, fine-focus source and a nickel filter. Thediffractometer was configured using the symmetric Bragg-Brentanogeometry. Prior to the analysis, a silicon specimen (NIST SRM 640e) wasanalyzed to verify the observed position of the Si 111 peak isconsistent with the NIST-certified position. A specimen of the samplewas prepared as a thin, circular layer centered on a siliconzero-background substrate. Antiscatter slits (SS) were used to minimizethe background generated by air. Soller slits for the incident anddiffracted beams were used to minimize broadening from axial divergence.Diffraction patterns were collected using a scanning position-sensitivedetector (X′Celerator) located 240 mm from the sample and Data Collectorsoftware v. 2.2b.

HPLC Procedures

The following table presents parameters and conditions for HPLCmeasurements described herein. Reported HPLC purities of 4a and 4b donot take into account a peak for proline.

Column Thames Restek Raptor C18 150 × 4.6 mm, 2.7 μm Mobile phase AWater Mobile phase B Acetonitrile Flow rate 1.0 mL/min UV Wavelength 215nm Column Temperature 40° C. Injection Volume 10 μL Runtime 40 minutesGradient Time (mins) Mobile Phase A (%) Mobile Phase B (%) 0 90 10 3 9010 33 10 90 35 10 90 37 90 10 40 90 10

Example 1: Synthesis of 4a/L-Proline Co-Crystal (Material A)

Compound 4a was obtained by chromatographic separation (HPLC: 4a 96.4%and 4b 1.2%, 500 mg) and was mixed with L-proline (128 mg, 1 eq.) inEtOH (4 mL). The mixture was heated at reflux for 15 min. The hotsolution was filtered through a cotton plug. The resulting clearfiltrate was cooled slowly and kept at room temperature for 16 h. Theprecipitated solid was collected by filtration, and dried in air at roomtemperature to give a 4a/L-proline co-crystal that was designated asMaterial A (456 mg, 73% yield) as a white solid. M.P. 203-205° C. ¹H NMRindicated a ratio of 4a to L-proline as 1:1.1.

Material A is a 1:1 4a/L-proline cocrystal and it is likely anisostructural solvate. Material A contains a minor L-proline componentbased on the XRPD pattern (FIG. 23 ), which was successfully indexed(Table A1). XRPD indexing is typically successful for samples consistingprimarily or exclusively of a single crystalline phase. However, anindexing solution was obtained for this mixture with the understandingthat the minor peaks/shoulders present in the XRPD pattern at 8.7°,15.0°, and 18.0° 20 are not consistent with the indexing solution andare likely attributable to L-proline. Select unit cell parametersobtained from the indexing solution are presented in Table A2.

TABLE A1 Observed peaks for 4a/L-proline Material A °2θ d space (Å)Intensity (%)  5.81 ± 0.20 15.189 ± 0.522   6  8.52 ± 0.20 10.369 ±0.243  100  9.19 ± 0.20 9.613 ± 0.209  63  9.92 ± 0.20 8.913 ± 0.179  2310.49 ± 0.20 8.426 ± 0.160  7 11.68 ± 0.20 7.568 ± 0.129  57 11.87 ±0.20 7.451 ± 0.125  65 12.21 ± 0.20 7.244 ± 0.118  29 13.22 ± 0.20 6.691± 0.101  67 14.75 ± 0.20 5.999 ± 0.081  73 16.07 ± 0.20 5.510 ± 0.068 18 16.33 ± 0.20 5.422 ± 0.066  78 16.68 ± 0.20 5.312 ± 0.063  20 17.10± 0.20 5.181 ± 0.060  45 17.57 ± 0.20 5.044 ± 0.057  54 18.32 ± 0.204.839 ± 0.052  29 18.54 ± 0.20 4.783 ± 0.051  27 18.87 ± 0.20 4.698 ±0.049  30 19.26 ± 0.20 4.605 ± 0.047  39 19.50 ± 0.20 4.548 ± 0.046  7519.65 ± 0.20 4.514 ± 0.045  35 20.17 ± 0.20 4.399 ± 0.043  27 20.34 ±0.20 4.363 ± 0.042  46 21.22 ± 0.20 4.183 ± 0.039  70 21.79 ± 0.20 4.076± 0.037  21 22.08 ± 0.20 4.023 ± 0.036  8 22.34 ± 0.20 3.976 ± 0.035  822.61 ± 0.20 3.930 ± 0.034  6 23.51 ± 0.20 3.781 ± 0.032  35 23.79 ±0.20 3.738 ± 0.031  50 24.58 ± 0.20 3.619 ± 0.029  26 24.88 ± 0.20 3.576± 0.028  15 25.12 ± 0.20 3.542 ± 0.028  13 25.48 ± 0.20 3.493 ± 0.027 24 25.96 ± 0.20 3.430 ± 0.026  8 26.20 ± 0.20 3.398 ± 0.025  16 26.45 ±0.20 3.367 ± 0.025  9 27.28 ± 0.20 3.266 ± 0.023  6 27.63 ± 0.20 3.225 ±0.023  14 27.85 ± 0.20 3.201 ± 0.023  11 28.27 ± 0.20 3.154 ± 0.022  1128.40 ± 0.20 3.140 ± 0.022  14 29.04 ± 0.20 3.072 ± 0.021  6 29.46 ±0.20 3.029 ± 0.020  5 29.78 ± 0.20 2.998 ± 0.020  14

TABLE A2 Unit Cell Parameters for 4a/L-proline Material A Bravais TypePrimitive Orthorhombic a [Å] 10.126 b [Å] 11.021 c [Å] 30.259 α [deg] 90β [deg] 90 γ [deg] 90 Volume [Å³/cell] 3,376.9 Chiral Contents? ChiralExtinction Symbol P 2₁ 2₁ - Space Group(s) P2₁2₁2 (18)

The unit cell volume is large enough to accommodate a solvated 1:14a/L-proline cocrystal. The free volume (or the unit cell volumeremaining after the cocrystal is accounted for) could possibly fit waterand/or any of the solvents from which Material A was produced, includingEtOH, IPA, and THF.

Material A as described above was additionally characterized by DSC,TGA, and DVS. An overlay of DSC and TGA thermograms for the material isshown in FIG. 24 . The TGA thermogram exhibits two distinct weight losssteps, the first occurring between −100 and 160° C. (7 wt %) and thesecond between 160 and 230° C. (21 wt %). A broad endotherm is observedby DSC with a peak maximum at 145° C., which corresponds to the firstTGA weight loss step. The relatively high temperatures at which theseevents occur as well as the stepwise nature of the weight loss likelyindicate the loss of bound solvent/water. Overlapping endothermic eventsbetween ˜170 and ˜240° C. by DSC correspond to the second weight lossstep in the TGA thermogram, likely corresponding with concurrent meltingof the cocrystal and volatilization of the L-proline component. Thesteep drop in the TGA thermogram above ˜250° C. likely corresponds withdecomposition.

A DVS isotherm for Material A as described above is presented in FIG. 25. Because the material was characterized as a mixture with unreactedL-proline, it is unknown what effect, if any, the excess L-proline mighthave had on the vapor sorption behavior. The material exhibitssignificant hygroscopicity at or above 85% RH, taking up ˜6 wt % watervapor between 85% and 95% RH. The vapor sorption kinetic equilibrationtimed out at 85%-95% RH, indicating that the cocrystal could potentiallypick up more moisture than what was measured if it was allowed a longerequilibration time. Relatively steady weight loss was noted upondesorption between 95% and 5% RH. The weight lost upon desorption (˜8 wt%) was significantly higher than that gained during sorption, indicatingthe material was likely solvated/hydrated at the start of the analysis.Analysis of the post-DVS material by XRPD showed a decrease incrystallinity, although the solid form remained intact. The presence orabsence of excess L-proline in the post-DVS sample could not beconfirmed due to the disorder in the XRPD pattern.

Example 2: Purification of 4a by Co-Crystallization with L-Proline

A 500 mg mixture of 4 consisting of compounds 4a and 4b (HPLC: 92.0% of4a and 7.1% 4b) and L-proline (128 mg, 1 eq) in ethanol (4 mL) wasrefluxed for 15 min. The mixture was seeded with a co-crystal obtainedin Example 1, and the mixture was allowed to cool and then kept at roomtemperature for 18 h. A white solid was filtered off, while residualsolid was transferred out of the reaction flask with the mother liquor.The collected quantity of 4a/L-proline co-crystal (1:1 ratio asdetermined by ¹H-NMR, 497 mg, 79% yield) consisted of 98.0% 4a and 1.49%4b (analyzed by HPLC).

Example 3: Co-Crystal Screen

Amorphous 4a was utilized in approximately 50 co-crystal screenexperiments with 26 co-formers other than L- and D-proline, assummarized in Table 1 below. A variety of crystallization techniquesamenable to co-crystal formation was employed, includingsolvent-assisted milling and manual grinding, cooling, evaporation,slurry, crash precipitation, and reaction crystallization, in which asolution containing a high molar excess of one component is combinedwith another component to encourage the reaction equilibrium to favorco-crystal formation. A variety of coformers possessing functionalgroups capable of forming hydrogen bonds was utilized, includingcarboxylic acids, amino acids, sugars, amides, amines, and numerousfunctional aromatic compounds. Under the variety of conditions andcoformers explored in this screen, however, 4a did not form anyconfirmed co-crystals with these common coformers.

TABLE 1 4a/Coformer Coformer Molar Ratio Conditions Technique Resultsacetic acid ~1:66 1) add glacial acetic acid to Obs 1) solids turnedlight blue and 4a solids w/ stirring then dissolved, clear light 2) RC(stir), RT, 3 days bluish soln. 3) ref., 73 days 2) clear soln. 3) clearsoln. L-arginine 1:5 1) dissolve L-arginine in Obs 1) undissolved solidspresent water, add to 4a 2) clear liquid phase, off- 2) RC (stir), RT, 3days white gummy solids on stir 3) add 5 mol. eq. L-arginine bar 1:10 4)RC (stir), RT, 4 days 3) undissolved solids present 4) clear liquidphase, gummy solids stuck to stir bar L-arginine 1:1 1) mill 4a w/ MeOHat 30 Obs 1) sticky goo Hz for 3 × 10-min. cycles 2) sticky goo 2) dryunder N₂ 1 day caffeine 1:2 1) add MEK to 4a and Obs 1) clear soln.coformer solids w/ stirring 2) large mass of off-white at ~74° C.solids, small amt. liquid 2) SC, ~74° C. to RT, stand visible at RT, 1day 3) liquid released from solids 3) poke w/ spatula 4) white solids 4)vac. filter OM needles, B/E XRPD caffeine caffeine 1:1 manually grind 4aw/ Obs free-flowing off-white solids acetone 4 min. OM fines andaggregates, partial B/E XRPD caffeine + amorphous carbamazepine 1:1 1)add EtOAc to 4a and Obs 1) clear soln. coformer solids w/ stirring 2)opaque white suspension, at ~75° C. white solids on walls 2) SC, ~75° C.to RT, stir at 3) white solids RT 3 days OM fines and aggregates, B/E 3)vac. filter XRPD carbamazepine citric acid 1:5 1) dissolve citric acidin Obs 1) clear soln. EtOH, add to 4a 2) clear soln. 2) RC (stir), RT, 3days 3) clear soln. 3) add 5 mol. eq. citric acid 4) clear soln. 1:10 4)RC (stir), RT, 4 days 5) clear soln. 5) add 10 mol. eq. citric 6) clearsoln. acid 1:20 6) RC (stir), RT, 46 days D-fructose 1:5 1) dissolveD-fructose in Obs 1) clear soln. MeOH, add to 4a 2) clear soln. 2) RC(stir), RT, 3 days 3) undissolved solids present 3) add 5 mol. eq.fructose 4) clear soln. 1:10 4) RC (stir), RT, 4 days 5) undissolvedsolids present 5) add 10 mol. eq. fructose 6) clear liquid phase, white1:20 6) RC (stir), RT, 16 days solids 7) vac. filter 7) white solids OMfines and aggregates, B/E XRPD D-fructose fumaric acid 1:2 1) add EtOHto 4a and Obs 1) clear soln. acid solids w/ stirring at 2) cloudy whitesuspension ~74° C. 3) white solids 2) SC, ~74° C. to RT, stir at OMfines and aggregates, B/E RT 1 day XRPD fumaric acid 3) vac. filterfumaric acid 1:10 1) dissolve acid in THF, Obs 1) clear soln. add to 4a2) clear soln. 2) RC (stir), RT, 3 days 3) undissolved solids present 3)add 10 mol. eq. fumaric 4) clear liquid phase, white acid solids 1:20 4)RC (stir), RT, 12 days 5) white solids 5) vac. filter OM fines andaggregates, B/E XRPD fumaric acid gentisic acid 1:3 1) dissolve 4a andacid in Obs 1) clear soln. ACN 2) clear soln. 2) stir, RT, 1 day 3)soln. became very slightly 3) add MTBE w/ stirring cloudy, then cleared(ACN/MTBE 1:3) 4) clear, slightly amber soln. 4) stir, RT, 3 days 5)large crystals embedded in 5) SE sticky amber oil 6) vac. oven, RT, 3days 6) bubbly off-white solids OM (after step numerous plates (likely5) singles), B/E; oil, no B/E OM (after step unknown morphology, no 6)B/E; plates, B/E XRPD gentisic acid L-glutamic acid 1:2 1) add EtOH andwater Obs 1) slightly hazy soln. (1:2) to 4a w/ stirring 2) clear soln.at ~75° C. 3) clear soln. 2) hot filter 4) clear liquid phase, white 3)SC, ~75° C. to RT, stand solids on bottom at RT 4 days 5) damp whitesolids 4) ref., 6 days OM irregular plates and 5) decant liquid, drysolids agglomerates, B/E briefly under N₂ XRPD L-glutamic acid glycine1:2 1) dissolve 4a in EtOH Obs 1) clear soln. 2) add 4a soln. to glycine2) undissolved solids present 3) slurry, RT, 4 days 3) cloudysuspension, large 4) add 4a to 1:1 crystals on bottom (likely 1:1 5)stir, RT, 3 days glycine) 6) add 4a to 1.5:1 4) cloudy 1.5:1 7) stir,RT, days 5) cloudy suspension, few 8) vac. filter large crystals (likelyglycine) present 6) cloudy 7) cloudy suspension, minimal large crystalson bottom 8) white solids OM fines, aggregates, and tablets, B/E XRPDglycine glycine 1:2 1) mill 4a w/ toluene at 30 Obs 1) sticky goo andwhite Hz for 3 × 10-min. cycles solids 2) dry under N₂ 1 day 2)off-white sticky goo hippuric acid 1:1 1) add MEK to 4a and Obs 1) clearsoln. acid solids w/ stirring at 2) clear liquid phase, white ~74° C.solids coating bottom 2) SC, ~74° C. to RT, stand 3) white solids at RT,1 day OM rectangular plates and 3) decant liquid, dry solids aggregates,B/E briefly under N₂ XRPD hippuric acid trans-4- 5:1 1) dissolve 4a inEtOH Obs 1) slightly hazy light yellow hydroxy-L- 2) add 4a soln. tosoln. proline coformer solids w/ stirring 2) small amt. undissolved 3)RC (stir), RT, 6 days solids present 4) vac. filter 3) opaque off-whitesuspension 4) white solids OM fines and aggregates, no B/E XRPDtrans-4-hydroxy-L-proline D-(-)-isoascorbic 1:2 1) add EtOH to 4a andObs 1) clear soln. acid acid solids w/ stirring at 2) opaque whitesuspension, ~75° C. white solids on walls 2) SC, ~75° C. to RT, stir at3) white solids RT 3 days OM fines and aggregates, B/E 3) vac. filterXRPD D-isoascorbic acid lactic acid ~1:48 1) add conc. lactic acid toObs 1) thick suspension, 4a solids w/ stirring undissolved solidspresent 2) RC (stir), RT, 3 days 2) clear soln. 3) ref., 73 days 3)clear soln. nicotinamide 1:3 1) dissolve 4a and acid in Obs 1) clearsoln. MEK 2) clear soln. 2) stir, RT, 1 day 3) small amt. clear liquid3) partial SE phase, off-white solids on 4) decant liquid, dry solidsbottom and sides briefly under N₂ 4) sticky white solids OM thinneedles, B/E XRPD nicotinamide nicotinamide 1:20 1) dissolvenicotinamide in Obs 1) clear soln. MeOH, add to 4a 2) clear liquidphase, white 2) RC (stir), RT, 1 day solids present 3) vac. filter 3)white solids OM needles and aggregates, B/E XRPD nicotinamide oxalicacid 1:10 1) add ACN to 4a and Obs 1) slightly hazy soln. acid solids w/sonication 2) clear soln. 2) RC (stir), RT, 2 days 3) undissolved solidspresent 3) add 10 mol. eq. acid 4) clear amber liquid phase, 1:20 4) RC(stir), RT, 11days white solids present 5) decant liquid phase, dry 5)white solids solids under N2 OM fines and aggregates, B/E XRPD oxalicacid L-phenylalanine 1:2 1) add EtOH to 4a and Obs 1) undissolved solidscoformer solids w/ stirring 2) slightly hazy suspension at ~73° C. 3)cloudy suspension 2) add water to EtOH/water (opaque) 50:50 4) whitesolids 3) SC, ~73° C. to RT, stir at OM fine needles and aggregates, RT1 day B/E 4) vac. filter XRPD L-phenylalanine hemihydrate piperazine1:1 1) manually grind Obs 1) sticky film 4a w/ acetone 4 min. 2) stickyfilm, could not be 2) dry under N₂ gas for scraped from mortar and 2min. pestle piracetam 1:2 1) add EtOH to 4a and Obs 1) clear soln.coformer solids w/ stirring 2) opaque off-white at ~75° C. suspension 2)SC, ~75° C. to RT, stir at 3) white solids RT 3 days OM fines andaggregates, B/E XRPD piracetam L-prolinamide 1:2 1) add EtOH to 4a andObs 1) clear yellow soln. coformer solids w/ stirring 2) clear yellowsoln. at ~75° C. 3) clear yellow soln. 2) SC, ~75° C. to RT, stir at 4)sticky yellow oil RT 3 days 5) clear liquid phase, yellow 3) freezer, 7days oil 4) SE 6) clear liquid phase, yellow 5) add diethyl ether oil onbottom 6) slurry (stir), RT, 22 days L-prolinamide 1:2 1) add ACN to 4aand Obs 1) clear yellow soln. coformer solids w/ stirring 2) clearyellow soln., at ~73° C. translucent film on walls 2) SC, ~73° C. to RT,stir at 3) yellow liquid phase, small RT 1 day amt. solids 3) freezer,16 days 4) stick yellow oil 4) SE 5) clear liquid, yellow oil 5) add IPEw/ stirring 6) clear liquid phase, yellow 6) stir, RT, 3 days oil onbottom propyl gallate 1:1 1) mill 4a w/ toluene at 30 Obs 1) sticky gooHz for 3 × 10-min. cycles 2) white sticky goo 2) dry under N₂ 1 daypropyl gallate 1:1 1) add EtOH to 4a w/ Obs 1) clear soln. stirring at~75° C. 2) clear soln. 2) SC, ~75° C. to RT, stand 3) clear soln. at RT4 days 4) clear soln. 3) freezer, 2 days 5) clear soln. 4) partial SE 6)sticky goo 5) freezer (capped), 3 days 7) clear soln. 6) SE 8) clearsoln. 7) add MTBE w/ stirring 9) sticky amber oil 8) stir, RT, 30 days9) SE pyrazine 1:5 1) dissolve pyrazine in Obs 1) clear soln. acetone,add to 4a 2) clear yellow soln. 2) RC (stir), RT, 3 days 3) clear soln.3) add 5 mol. eq. pyrazine 4) clear soln. 1:10 4) RC (stir), RT, 4 days5) clear light yellow soln. 5) add 10 mol. eq. pyrazine 6) clear soln.1:20 6) RC (stir), RT, 46 days pyrazine 1:2 1) add EtOH to 4a w/ Obs 1)clear soln. stirring at ~75° C. 2) clear soln. 2) SC, ~75° C. to RT,stand 3) clear soln. at RT 4 days 4) clear soln. 3) freezer, 2 days 5)clear soln. 4) partial SE 6) sticky goo 5) freezer (capped), 3 days 7)clear liquid phase, light 6) SE yellow goo 7) add heptane w/ stirring 8)clear liquid phase, oil on 8) stir, RT, 30 days bottom L-pyroglutamic1:1 1) mill 4a w/ MeOH at 30 Obs 1) sticky goo acid Hz for 3 × 10-min.cycles 2) sticky goo 2) dry under N₂ 1 day L-pyroglutamic 1:2 1) addEtOH to 4a w/ Obs 1) clear soln. acid stirring at ~75° C. 2) clear soln.2) SC, ~75° C. to RT, stand 3) clear soln. at RT 4 days 4) clear soln.3) freezer, 2 days 5) clear soln. 4) partial SE 6) sticky goo 5) freezer(capped), 3 days 7) clear liquid phase, white 6) SE goo 7) add diethylether w/ 8) clear liquid phase, white stirring solids 8) stir, RT, 5days 9) white solids 9) decant liquid phase, dry OM fines andaggregates, B/E solids under N₂ XRPD Pyroglutamic Material A +pyroglutamic acid L-pyroglutamic 1:1 1) add EtOH to 4a and Obs 1) clearsoln. acid acid solids w/ sonication 2) small amt. clear soln. 2)partial FE, 1 day 3) clear viscous oil 3) evaporate under stream 4) oilbecame white of N₂ 5) clear liquid phase, white 4) add diethyl ether w/solids present stirring 6) white solids 5) add seedsª, stir, RT, OMfines and aggregates, B/E 10 days XRPD Pyroglutamic Material B + 6) vac.filter pyroglutamic acid L-pyroglutamic 1:1 1) add EtOH to 4a andObs/OM 1) clear soln. acid acid solids w/ sonication 2) seeds alwaysdissolved, 2) alternatively add always clear soln. seedsª and aliquots3) clear soln. diethyl ether multiple 4) clear soln. times w/ stirringto 5) sticky oil, small amt. ether/EtOH 6:1 ratio solids on upper walls3) stir, RT, 1 day (irregular plates, B/E) 4) stir, 2-8° C., 11 days 6)clear soln., oil on bottom 5) SE 7) clear liquid phase, off- 6) scrapesolids down to white solids oil, add diethyl ether w/ 8) off-whitesolids stirring OM fines and aggregates, B/E 7) stir, RT, 1 day (afterstep 8) 8) decant liquid, dry solids XRPD Pyroglutamic Material A +briefly under N₂ Material B + acid L-pyroglutamic 2:1 1) add EtOH to 4aand Obs 1) clear soln., few floats acid solids w/ sonication 2) clearsoln. 2) filter 3) clear viscous oil 3) evaporate under N₂ 4) seedsremained stream 5) oil turned white, clear 4) add seeds^(b) liquid phase5) add diethyl ether w/ 6) clear liquid phase, white stirring solidsacid 6) stir, RT, 18 days 7) white solids 7) vac. filter OM fines andaggregates, B/E XRPD Pyroglutamic Material B ± pyroglutamic acid2,3,5,6- 1:10 1) add EtOAc to 4a and Obs 1) clear soln. tetramethyl-coformer solids w/ 2) clear soln. pyrazine (TMP) sonication 3)undissolved solids present 2) RC (stir), RT, 2 days 4) clear soln. 3)add 10 mol eq. TMP 1:20 4) RC (stir), RT, 36 days L-tryptophan 1:2 1)add EtOH to 4a and Obs 1) undissolved solids present coformer solids w/stirring 2) clear soln. at ~73° C. 3) clear liquid phase, white 2) addwater to EtOH/water solids 3:1 4) shiny white solids (like a 3) SC, ~73°C. to RT, stir at pearl) RT 1 day OM aggregates, B/E 4) vac. filter XRPDL-tryptophan urea 1:2 1) add EtOH to 4a and Obs 1) clear soln. ureasolids w/ stirring at 2) clear soln. ~74° C. 3) slightly hazy suspension2) SC, ~74° C. to RT, stir at 4) slightly hazy suspension RT 1 day 5)clear liquid phase, small 3) freezer, 3 days amt. solids 4) add EtOAc w/stirring 6) clear liquid phase, small (EtOAc/EtOH 6:1) amt. white solids5) stir, RT, 1 day 7) white solids 6) freezer, 7 days OM needles, B/E 7)decant liquid phase, dry XRPD urea solids briefly under N₂ ^(a,b)Variousbatches of seeds of uncharacterized crystalline material comprising 4aand pyroglutamic acid.

Example 4: Preparation and Characterization of 4a/L-Proline Form B

Equimolar amounts of amorphous 4a and L-proline (1:1) were mixed inmethanol and were heated to about 68° C. The resulting solution wasallowed to slowly cool to room temperature, at which point a whitesuspension had formed. The suspension was then stirred at roomtemperature for three days, after which Form B as a white solid wascollected by filtration and dried.

Alternatively, amorphous 4a and L-proline in a 1:2 molar ratio werecombined in ethanol and heated to about 82° C. to yield a whitesuspension. The suspension was held at 82° C. for about 5 minutes,allowed to slowly cool to room temperature, and then stirred at roomtemperature for three days. Form B was collected as a white solid byfiltration and dried.

Form B is an anhydrous 1:2 4a/L-proline co-crystal. Form B wascharacterized by XRPD (with indexing), DSC, TGA, DVS, Ramanspectroscopy, IR spectroscopy, proton NMR, HPLC and elemental analysis.

The XRPD pattern for Form B was successfully indexed (Table 2) and itindicated that Form B consists primarily or exclusively of a singlecrystalline phase (FIG. 1 ). The unit cell volume obtained from theindexing solution is consistent with an anhydrous 1:2 4a/L-prolineco-crystal. Table 3 below presents unit cell parameters.

TABLE 2 Observed peaks for 4a/L-proline Form B °2θ d space (Å) Intensity(%)  5.81 ± 0.20 15.204 ± 0.523  36  8.40 ± 0.20 10.512 ± 0.250  21 8.50 ± 0.20 10.396 ± 0.244  16 10.46 ± 0.20 8.454 ± 0.161 29 11.65 ±0.20 7.590 ± 0.130 15 12.14 ± 0.20 7.286 ± 0.120 30 14.57 ± 0.20 6.076 ±0.083 43 14.76 ± 0.20 5.998 ± 0.081 71 16.49 ± 0.20 5.371 ± 0.065 1116.86 ± 0.20 5.253 ± 0.062 100  17.51 ± 0.20 5.061 ± 0.057 78 18.16 ±0.20 4.881 ± 0.053 21 18.39 ± 0.20 4.819 ± 0.052 25 18.89 ± 0.20 4.694 ±0.049 52 19.00 ± 0.20 4.667 ± 0.049 70 19.17 ± 0.20 4.627 ± 0.048 3019.41 ± 0.20 4.570 ± 0.047 61 19.58 ± 0.20 4.530 ± 0.046 27 19.93 ± 0.204.452 ± 0.044 14 21.05 ± 0.20 4.217 ± 0.040 47 21.48 ± 0.20 4.134 ±0.038 10 21.82 ± 0.20 4.070 ± 0.037 34 23.43 ± 0.20 3.794 ± 0.032 1923.56 ± 0.20 3.774 ± 0.032 29 23.77 ± 0.20 3.740 ± 0.031 24 24.36 ± 0.203.651 ± 0.030 44 25.13 ± 0.20 3.541 ± 0.028 17 25.71 ± 0.20 3.462 ±0.026 10 26.36 ± 0.20 3.378 ± 0.025 21 26.60 ± 0.20 3.348 ± 0.025 1626.94 ± 0.20 3.306 ± 0.024 14 27.18 ± 0.20 3.278 ± 0.024  9 27.48 ± 0.203.243 ± 0.023  9 27.67 ± 0.20 3.221± 0.023 14 27.97 ± 0.20 3.188 ± 0.02213 28.28 ± 0.20 3.153 ± 0.022  9 28.91 ± 0.20 3.086 ± 0.021 19 29.38 ±0.20 3.037 ± 0.020 12 29.75 ± 0.20 3.001 ± 0.020 10 29.99 ± 0.20 2.977 ±0.019  8

TABLE 3 Unit Cell Parameters for 4a/L-proline Form B Bravais TypePrimitive Monoclinic a [Å] 11.130 b [Å] 10.168 c [Å] 16.094 α [deg] 90 β[deg] 109.27 γ [deg] 90 Volume [Å³/cell] 1,719.3 Chiral Contents? ChiralExtinction Symbol P 1 2₁ 1 Space Group(s) P2₁ (4)

A sample of Form B isolated from a MeOH slurry was characterized byproton NMR and HPLC. The proton NMR data indicate a 1:2 4a/L-prolinestoichiometry with no residual solvent detected. The purity of 4a in thesample was 99.7% as determined by HPLC.

DSC and TGA thermograms for Form B are presented in FIG. 2 and FIG. 3 ,respectively. The data are plotted separately since different sampleswere analyzed for each technique. The sample analyzed by TGA wasisolated from a MeOH slurry, while the DSC sample resulted from aco-crystal formation experiment in MeOH. Virtually no weight loss isobserved by TGA between ambient temperature and 190° C., consistent withan anhydrous/non-solvated material. The DSC is consistent with this aswell, showing no notable thermal events until the onset of anendothermic event at 208° C., with an overlapping strong exothermicevent. To be noted, the sample was observed to come out of the panfollowing this analysis, likely contributing to the magnitude of theexotherm. These events likely correspond with the melting/dissociationof the co-crystal. Similarly, a steep decrease in the TGA thermogramabove 190° C. is likely attributed to volatilization of a portion of theL-proline component of the co-crystal, followed by likely decomposition.

To further confirm the co-crystal stoichiometry, Form B was analyzed byC, H, N, F, and O elemental analyses (Table 4). Comparison of theexperimental percent composition values to theoretical values for a 1:1and 1:2 co-crystal show that the sample is more closely consistent witha 1:2 co-crystal. This result is consistent with the othercharacterization data.

TABLE 4 Elemental Analysis of 4a/L-proline Form B Theoretical 1:1Theoretical 1:2 Experimental co-crystal co-crystal Results 61.9% C 60.3% C  59.78% C  7.0% H 7.1% H 6.88% H 5.0% N 6.2% N 6.28% N 3.4% F 2.8% F  2.79% F  22.7% O  23.6% O  25.12% O 

A dynamic vapor sorption (DVS) isotherm for Form B is shown in FIG. 4 .Weight gain of 2.3 wt % was noted between 5% and 95% RH, with themajority of the sorption occurring above 50% RH. All of this weight waslost on desorption with minor hysteresis noted. The vapor sorptionkinetic equilibration timed out on the sorption step between 85%-95% RH,indicating that the co-crystal could potentially pick up more moisturethan what was measured if it was allowed a longer equilibration time.Analysis of the post-DVS material by XRPD showed no observable change inform.

IR and Raman spectra were acquired for Form B and are presented in FIG.5 and FIG. 6 , respectively.

Example 5: Single Crystal X-Ray Structure Determination of 4a/L-ProlineForm B

Data Collection

A colorless plate of 4a/L-proline Form B (C₃₄H₄₈FN₃O₁₀ [C₂₄H₃₀FNO₆,2(C₅H₉NO₂)]) having approximate dimensions of 0.70×0.45×0.30 mm, wasmounted on a nylon loop in random orientation. Preliminary examinationand data collection were performed with Cu Kα radiation (λ=1.54178 Å) ona Rigaku Rapid II diffractometer equipped with confocal optics.Refinements were performed using SHELX2013 (Sheldrick, G. M. ActaCryst., 2008, A64, 112).

Cell constants and an orientation matrix for data collection wereobtained from least-squares refinement using the setting angles of 21646reflections in the range 4°<θ<68°. The refined mosaicity fromDENZO/SCALEPACK was 0.44° indicating good crystal quality (Otwinowski,Z.; Minor, W. Methods Enzymol. 1997, 276, 307). The space group wasdetermined by the program XPREP (Bruker, XPREP in SHELXTL v. 6.12.,Bruker AXS Inc., Madison, WI, USA, 2002). From the systematic presenceof the following conditions: 0k0 k=2n, and from subsequent least-squaresrefinement, the space group was determined to be P21 (no. 4).

The data were collected to a maximum diffraction angle (29) of 135.73°,at room temperature.

Data Reduction

Frames were integrated with HKL3000 (Otwinowski (1997)). A total of21646 reflections were collected, of which 5936 were unique. Lorentz andpolarization corrections were applied to the data. The linear absorptioncoefficient is 0.833 mm⁻¹ for Cu Kα radiation. An empirical absorptioncorrection using SCALEPACK (Otwinowski (1997)) was applied. Transmissioncoefficients ranged from 0.128 to 0.779. A secondary extinctioncorrection was applied (Sheldrick (2008)). The final coefficient,refined in least-squares, was 0.0157(11) (in absolute units).Intensities of equivalent reflections were averaged. The agreementfactor for the averaging was 4.1% based on intensity.

Structure Solution and Refinement

The structure was solved by direct methods using SHELXT (Sheldrick(2008)). The remaining atoms were located in succeeding differenceFourier syntheses. Hydrogen atoms were included in the refinement butrestrained to ride on the atom to which they are bonded. The structurewas refined in full-matrix least-squares by minimizing the function:

Σw(|F _(o)|² −|F _(c)|²)²

The weight w is defined as 1/[σ² (F_(o) ²)+(0.0640P)²+(0.5095P)], whereP=(F_(o) ²+2F_(c) ²)/3.

Scattering factors were taken from the “International Tables forCrystallography” (International Tables for Crystallography, Vol. C,Kluwer Academic Publishers: Dordrecht, The Netherlands, 1992, Tables4.2.6.8 and 6.1.1.4). Of the 5936 reflections used in the refinements,only the reflections with F_(o) ²>2σ(F_(o) ²) were used in calculatingthe fit residual, R. A total of 5601 reflections were used in thecalculation. The final cycle of refinement included 490 variableparameters and converged (largest parameter shift was <0.01 times itsestimated standard deviation) with unweighted and weighted agreementfactors of:

R=Σ|F _(o) −F _(c) |/ΣF _(o)=0.0422

R _(w)=√{square root over ((Σw(F _(o) ² −F _(c) ²)² /Σw(F _(o)²)²))}=0.1141

The standard deviation of an observation of unit weight (goodness offit) was 1.062. The highest peak in the final difference Fourier had aheight of 0.187 e/Å³. The minimum negative peak had a height of −0.193e/Å³.

Calculated X-Ray Powder Diffraction (XRPD) Pattern

A calculated XRPD pattern was generated for Cu radiation using Mercury(Macrae, C. F. Edgington, P. R. McCabe, P. Pidcock, E. Shields, G. P.Taylor, R. Towler M. and van de Streek, J.; J. Appl. Cryst., 2006, 39,453-457) and the atomic coordinates, space group, and unit cellparameters from the single crystal structure.

Atomic Displacement Ellipsoid and Packing Diagrams

The atomic displacement ellipsoid diagram was prepared using Mercury(Macrae (2006)). Atoms are represented by 50% probability anisotropicthermal ellipsoids. Packing diagrams and additional figures weregenerated with Mercury (Macrae (2006)). Hydrogen bonding is representedas dashed lines. Assessment of chiral centers was performed with PLATON(Spek, A. L. PLATON. Molecular Graphics Program. Utrecht University,Utrecht, The Netherlands, 2008. Spek, A. L, J. Appl. Cryst. 2003, 36,7). Absolute configuration is evaluated using the specification ofmolecular chirality rules (Cahn, R. S.; Ingold, C; Prelog, V. Angew.Chem. Intern. Ed. Eng., 1966, 5, 385; Prelog, V., Helmchen, G. Angew.Chem. Intern. Ed. Eng., 1982, 21, 567).

Results

The monoclinic cell parameters and calculated volume are: a=11.1270(4)Å, b=10.1566(4) Å, c=16.0790(6) Å, β=109.309(2)° (α=γ=90°),V=1714.91(11) Å³. The formula weight of the asymmetric unit in thecrystal structure of Form B is 677.75 g mol⁻¹ with Z=2, resulting in acalculated density of 1.313 g cm⁻³. The space group was determined to beP2₁ (no. 4). A summary of the crystal data and crystallographic datacollection parameters are provided in Table 5.

TABLE 5 Crystal Data and Data Collection Parameters for 4a/L-prolineForm B formula C₃₄H₄₈FN₃O₁₀ formula weight 677.77 space group P2₁ (No.4) a, Å 11.1270(4) b, Å 10.1566(4) c, Å 16.0790(6) b, deg 109.309(2) V,Å³ 1714.91(11) Z 2 d_(calc), g cm⁻³ 1.312 crystal dimensions, mm 0.25 ×0.20 × 0.16 temperature, K 295 radiation (wavelength, Å) Cu K_(a)(1.54178) monochromator confocal optics linear abs coef, mm⁻¹ 0.833absorption correction applied empirical^(a) transmission factors: min,max 0.128, 0.779 diffractometer Rigaku RAPID-II h, k, l range −13 to 13−12 to 12 −18 to 18 2q range, deg 8.42-135.73 mosaicity, deg 0.44programs used SHELXTL F₀₀₀ 724 data collected 21646 unique data 5936R_(int) 0.041 data used in refinement 5936 cutoff used in R-factor F_(o)² > 2.0 s(F_(o) ²) calculations data with I > 2.0 s(I) 5601 refinedextinction coef 0.0157 number of variables 490 largest shift/esd infinal cycle 0 R(F_(o)) 0.0422 R_(w)(F_(o) ²) 0.1141 goodness of fit1.062 absolute structure Flack parameter^(b) (−0.02(11)) determinationHooft parameter^(c) (−0.02(5)) Friedel Coverage 92% ^(a)Otwinowski, Z.;Minor, W. Methods Enzymol. 1997, 276, 307. ^(b)Flack, H. D. Acta Cryst.,1983 A39, 876. ^(c)Hooft, R. W. W., Straver, L. H., and Spek, A. L. J.Appl. Cryst., 2008, 41, 96-103.

The space group and unit cell parameters are consistent with thoseobtained from XRPD analysis of Form B (see Table 3 above).

The quality of the structure obtained is high, as indicated by the fitresidual, R of 0.0422 (4.22%). R-values in the range of 0.02 to 0.06 arequoted for the most reliably determined structures (Glusker, JennyPickworth; Trueblood, Kenneth N. Crystal Structure Analysis: A Primer,2^(nd) ed.; Oxford University press: New York, 1985; p. 87).

An atomic displacement ellipsoid drawing of Form B is shown in FIG. 7 .The molecule observed in the asymmetric unit of the single crystalstructure is consistent with the proposed molecular structure of 4a. Theasymmetric unit shown in FIG. 7 contains one 4a molecule and twoL-proline molecules, consistent with a 1:2 4a: L-proline stoichiometry.Two protons were located and refined independently on both of theproline nitrogen atoms, indicating zwitterions. Both of the L-prolinemolecules are disordered over two positions, refining to 82/18% and71/29% occupancies.

The absolute structure can be determined through an analysis ofanomalous X-ray scattering by the crystal. A refined parameter x, knownas the Flack parameter (Flack, H. D.; Bernardinelli, G., Acta Cryst.1999, A55, 908; Flack, H. D.; Bernardinelli, G., J. Appl. Cryst. 2000,33, 1143; Flack, H. D. Acta Cryst. 1983, A39, 876; Parsons, S., Flack,H. D., Wagner, T., Acta Cryst. 2013, B69, 249-259), encodes the relativeabundance of the two components in an inversion twin. The structurecontains a fraction 1-x of the model being refined, and x of itsinverse. Provided that a low standard uncertainty is obtained, the Flackparameter should be close to 0 if the solved structure is correct, andclose to 1 if the inverse model is correct. The measured Flack parameterfor the structure of Form B shown in FIG. 7 is −0.02 with a standarduncertainty of 0.11, which indicates weak inversion-distinguishingpower, and therefore no interpretation of the Flack parameter could bemade. The error in the standard uncertainty prevents an assignment basedsolely on the Flack factor.

Refinement of the Flack parameter (x) does not result in a quantitativestatement about the absolute structure assignment. However, an approachapplying Bayesian statistics to Bijvoet differences can provide a seriesof probabilities for different hypotheses of the absolute structure(Hooft, R. W. W., Straver, L. H., and Spek, A. L. J. Appl. Cryst., 2008,41, 96-103; Bijvoet, J. M.; Peerdeman, A. F.; van Bommel A. J. Nature1951, 168, 271). This analysis provides a Flack equivalent (Hooft)parameter in addition to probabilities that the absolute structure iseither correct, incorrect or a racemic twin. For the current data setthe Flack equivalent (Hooft) parameter was determined to be −0.02(5),the probability that the structure is correct is 1.000, the probabilitythat the structure is incorrect is 0.9×10⁻⁹¹ and the probability thatthe material is a racemic twin is 0.2×10⁻²⁴. Therefore, the absoluteconfiguration of the model in FIG. 7 is correct. This structure containsfour chiral centers on 4a located at C7, C9, C11, and C12 (FIG. 7 )which bond in the R,R,S, and R configuration, respectively, and onechiral center on each of the proline molecules at C26 and C31 bothbonding in the S configuration.

FIG. 8 shows a calculated XRPD pattern of Form B, generated from thesingle crystal structure. The previously indexed experimental XRPDpattern of Form B (Example 4) is shown above and is consistent with thecalculated XRPD pattern (FIG. 9 ).

Example 6: Preparation and Characterization of 4a/L-Proline Form C

Equimolar amounts of amorphous 4a and L-proline (1:1) were slurried andthen stirred in acetone at room temperature for 3 days. The slurry wasfiltered to collect Form C as a white solid.

Form C also was prepared by dissolving equimolar amounts of amorphous 4aand L-proline in EtOH. Acetone then was introduced to the solution byvapor diffusion (VD) to precipitate Form C.

The data indicated that Form C consists of a 1:1 co-crystal with 1 moleof acetone present in the crystal lattice, although the acetone does notparticipate in hydrogen bonding. The single crystal data providesconfirmation of chemical and solid phase compositions.

An XRPD pattern for Form C (FIG. 10 ) was successfully indexed, andobserved peaks are shown in Table 6.

TABLE 6 Observed peaks for 4a/L-proline Form C °2θ d space (Å) Intensity(%)  8.73 ± 0.20 10.126 ± 0.232  67 10.51 ± 0.20 8.414 ± 0.160 46 11.83± 0.20 7.477 ± 0.126 32 12.10 ± 0.20 7.308 ± 0.120 62 12.26 ± 0.20 7.214± 0.117 36 12.44 ± 0.20 7.110 ± 0.114 31 14.64 ± 0.20 6.047 ± 0.082 9715.14 ± 0.20 5.847 ± 0.077 50 16.13 ± 0.20 5.492 ± 0.068 13 17.53 ± 0.205.055 ± 0.057 100 18.26 ± 0.20 4.855 ± 0.053 62 18.91 ± 0.20 4.688 ±0.049 71 19.36 ± 0.20 4.580 ± 0.047 74 19.56 ± 0.20 4.536 ± 0.046 5620.17 ± 0.20 4.399 ± 0.043 20 20.97 ± 0.20 4.232 ± 0.040 24 21.15 ± 0.204.197 ± 0.039 22 21.33 ± 0.20 4.163 ± 0.039 54 21.55 ± 0.20 4.121 ±0.038 29 22.40 ± 0.20 3.966 ± 0.035 14 23.18 ± 0.20 3.834 ± 0.033 2923.71 ± 0.20 3.750 ± 0.031 20 24.02 ± 0.20 3.701 ± 0.030 33 24.34 ± 0.203.654 ± 0.030 25 24.73 ± 0.20 3.597 ± 0.029 40 25.87 ± 0.20 3.441 ±0.026 23 26.54 ± 0.20 3.356 ± 0.025 17 26.71 ± 0.20 3.335 ± 0.025 1527.09 ± 0.20 3.289 ± 0.024 12 27.38 ± 0.20 3.254 ± 0.023 16 27.86 ± 0.203.200 ± 0.023 8 28.40 ± 0.20 3.140 ± 0.022 12 28.73 ± 0.20 3.105 ± 0.02111 29.05 ± 0.20 3.071 ± 0.021 10 29.45 ± 0.20 3.031 ± 0.020 23

Unit cell parameters from XRPD indexing are presented in Table 7 below:

TABLE 7 Unit Cell Parameters for 4a/L-proline Form C Bravais TypePrimitive Monoclinic a [Å] 10.992 b [Å] 10.275 c [Å] 15.362 α [deg] 90 β[deg] 108.07 γ [deg] 90 Volume [Å³/cell] 1,649.5 Chiral Contents? ChiralExtinction Symbol P 1 2₁ 1 Space Group(s) P2₁ (4)

A TGA thermogram for Form C exhibits stepwise weight loss, consistentwith the finding that the material consists of an acetone solvate (FIG.11 ). The acetone appears to volatilize in two separate steps. In thefirst step, 2.6% weight loss is observed between ˜60 and 150° C. On theassumption that the volatile is acetone, the 2.6 wt % corresponds with0.26 mole (or ˜¼ of the total acetone per the single crystal structure).A second weight loss step between 150 and 220° C. corresponds with 6.7%weight loss, or 0.70 mol if acetone is assumed to be the only volatile.

Example 7: Single Crystal X-Ray Structure Determination of 4a/L-ProlineForm C

A colorless plate of C₃₂H₄₅FN₂O₉ [C₂₄H₃₀FNO₆, C₅H₉NO₂, C₃H₆O] havingapproximate dimensions of 0.19×0.18×0.10 mm, was mounted on a fiber inrandom orientation. Preliminary examination and data collection wereperformed with Cu K_(α), radiation (λ=1.54178 Å) on a Rigaku Rapid IIdiffractometer equipped with confocal optics. Refinements were performedusing SHELX2013 (Sheldrick (2008)).

Cell constants and an orientation matrix for data collection wereobtained from least-squares refinement using the setting angles of 12615reflections in the range 4°<θ<59°. The refined mosaicity fromDENZO/SCALEPACK was 0.25° indicating good crystal quality (Otwinowski(1997)). The space group was determined by the program XPREP (Bruker(2002)). From the systematic presence of the following conditions: 0k0k=2n, and from subsequent least-squares refinement, the space group wasdetermined to be P2₁ (no. 4).

The data were collected to a maximum diffraction angle (2θ) of 117.84°at room temperature.

Frames were integrated with HKL3000 (Bruker (2002)). A total of 12615reflections were collected, of which 4368 were unique. Lorentz andpolarization corrections were applied to the data. The linear absorptioncoefficient is 0.788 mm⁻¹ for Cu Kα radiation. An empirical absorptioncorrection using SCALEPACK (Bruker (2002)) was applied. Transmissioncoefficients ranged from 0.060 to 0.924. A secondary extinctioncorrection was applied (Sheldrick (2008)). The final coefficient,refined in least-squares, was 0.0049(7) (in absolute units). Intensitiesof equivalent reflections were averaged. The agreement factor for theaveraging was 4.8% based on intensity.

Structure solution and refinement were performed in a manner analogousto Example 5 above. Of the 4368 reflections used in the refinements,only the reflections with F_(o) ²>2 σ(F_(o) ²) were used in calculatingthe fit residual, R. A total of 3518 reflections were used in thecalculation. The final cycle of refinement included 417 variableparameters and converged (largest parameter shift was <0.01 times itsestimated standard deviation) with unweighted and weighted agreementfactors of:

R=Σ|F _(o) −F _(c) |/ΣF _(o)=0.0535

R _(w)=√{square root over ((Σw(F _(o) ² −F _(c) ²)² /Σw(F _(o)²)²))}=0.1305

The standard deviation of an observation of unit weight (goodness offit) was 1.078. The highest peak in the final difference Fourier had aheight of 0.271 e/Å³. The minimum negative peak had a height of −0.167e/Å³.

A calculated XRPD pattern and atomic displacement ellipsoid diagram weregenerated according to the procedure in Example 5.

The monoclinic cell parameters and calculated volume are: a=10.9962(6)Å, b=10.2721(6) Å, c=15.3197(9) Å, β=107.937(4)° (α=γ=90°),V=1646.32(17) Å³. The formula weight of the asymmetric unit in thecrystal structure of Form C is 620.70 g mol⁻¹ with Z=2, resulting in acalculated density of 1.252 g cm⁻³. The space group was determined to beP2₁ (no. 4). A summary of the crystal data and crystallographic datacollection parameters are provided in Table 8. The space group and unitcell parameters are in agreement with those obtained previously by XRPDindexing (Example 6).

TABLE 8 Crystal Data and Data Collection Parameters for 4a/L-prolineForm C formula C₃₂H₄₅FN₂O₉ formula weight 620.72 space group P2₁ (No. 4)a, Å 10.9962(6) b, Å 10.2721(6) c, Å 15.3197(9) b, deg 107.937(4) V, Å³1646.32(16) Z 2 d_(calc), g cm⁻³ 1.252 crystal dimensions, mm 0.19 ×0.18 × 0.15 temperature, K 293 radiation (wavelength, Å) Cu K_(a)(1.54178) monochromator confocal optics linear abs coef, mm⁻¹ 0.788absorption correction applied empirical^(a) transmission factors: min,0.79, 0.89 max diffractometer Rigaku RAPID-II h, k, l range −12 to 12−11 to 11 −17 to 17 2q range, deg 6.06-126.82 mosaicity, deg 0.25programs used SHELXTL F₀₀₀ 664 data collected 12615 unique data 4368R_(int) 0.048 data used in refinement 4368 cutoff used in R-factor F_(o)² > 2.0 s(F_(o) ²) calculations data with I > 2.0 s(I) 3518 refinedextinction coef 0.0049 number of variables 417 largest shift/esd infinal cycle 0 R(F_(o)) 0.054 R_(w)(F_(o) ²) 0.131 goodness of fit 1.078absolute structure Flack parameter^(b) (0.08(14)) determination Hooftparameter^(c) (0.09(9)) Friedel Coverage 86% ^(a)Otwinowski (1997).^(b)Flack (1983). ^(c)Hooft (2008).

An atomic displacement ellipsoid drawing of Form C is shown in FIG. 12 .The molecule observed in the asymmetric unit of the single crystalstructure is consistent with the proposed molecular structure of4a/L-proline. The asymmetric unit shown in FIG. 12 contains one 4amolecule, one L-proline molecule, and one acetone molecule. Two protonswere located and refined independently on the proline nitrogen atom,indicating a zwitterion.

FIG. 13 shows a calculated XRPD pattern of Form C, generated from thesingle crystal structure. The experimental XRPD pattern of the bulkmaterial from which the crystal was obtained is shown in FIG. 10 asdescribed above. All peaks in the experimental patterns are representedin the calculated XRPD pattern, indicating the bulk material is likely asingle phase. Differences in intensities between the calculated andexperimental powder diffraction patterns often are due to preferredorientation. Preferred orientation is the tendency for crystals to alignthemselves with some degree of order. This preferred orientation of thesample can significantly affect peak intensities, but not peakpositions, in the experimental powder diffraction pattern.

For the current data set the Flack equivalent (Hooft) parameter wasdetermined in the manner described in Example 5, and it was determinedto be 0.09(9), the probability that the structure is correct is 1.000,the probability that the structure is incorrect is 0.4×10⁻²¹ and theprobability that the material is a racemic twin is 0.4×10⁻⁴.

Therefore, the absolute configuration of the model in FIG. 12 is likelycorrect. This structure contains four chiral centers on 4a located atC7, C9, C11, and C12 (FIG. 12 ) which bond in the R, R, S, Rconfiguration, respectively and one chiral center on the proline locatedat C26 which bonds in the S configuration.

Example 8: Preparation and Characterization of 4a/L-Proline Form D

Equimolar amounts of amorphous 4a and L-proline were combined inacetonitrile to give a thin suspension, which was then heated to about85° C. The suspension was cooled to about 71° C., seeded with a smallquantity of material prepared in Example 1, and held at about 71° C. toabout 15 minutes. The suspension was then allowed to slowly cool to roomtemperature, and then stirred for three days. The resulting whitesuspension was filtered to yield Form D, which was then dried.

Form D was also prepared by combining equimolar amounts of amorphous 4aand L-proline in EtOH, heating to about 82° C. and held at thattemperature for about 15 minutes, cooling to about 76° C., seeding witha small quantity of material prepared in Example 1, slowly cooling totemperature, and stirring for three days. The resulting off-white slurrywas filtered, and the collected quantity of Form D was dried undervacuum at ˜48° C.

Form D consists of an anhydrous/non-solvated 1:2 4a/L-prolineco-crystal. An XRPD pattern for Form D was successfully indexed,indicating the material consists primarily or exclusively of a singlecrystalline phase (FIG. 14 , Table 9). The unit cell volume (Table 10)obtained from the indexing solution is consistent with an anhydrous 1:24a/L-proline co-crystal.

TABLE 9 Observed peaks for 4a/L-proline Form D °2θ d space (Å) Intensity(%)  2.82 ± 0.20 31.302 ± 2.219  13  5.68 ± 0.20 15.535 ± 0.546  50 8.45 ± 0.20 10.455 ± 0.247  16  9.20 ± 0.20 9.605 ± 0.208 100  9.78 ±0.20 9.038 ± 0.184 24 10.46 ± 0.20 8.454 ± 0.161 10 11.42 ± 0.20 7.741 ±0.135 25 11.83 ± 0.20 7.475 ± 0.126 62 12.17 ± 0.20 7.265 ± 0.119 3313.15 ± 0.20 6.729 ± 0.102 37 14.42 ± 0.20 6.138 ± 0.085 23 14.62 ± 0.206.054 ± 0.082 70 16.19 ± 0.20 5.470 ± 0.067 84 16.46 ± 0.20 5.382 ±0.065 16 16.94 ± 0.20 5.231 ± 0.061 47 17.16 ± 0.20 5.163 ± 0.060 8017.56 ± 0.20 5.047 ± 0.057 14 18.22 ± 0.20 4.864 ± 0.053 24 18.45 ± 0.204.804 ± 0.052 75 18.95 ± 0.20 4.680 ± 0.049 21 19.10 ± 0.20 4.643 ±0.048 30 19.31 ± 0.20 4.593 ± 0.047 46 19.52 ± 0.20 4.544 ± 0.046 7720.15 ± 0.20 4.403 ± 0.043 94 20.98 ± 0.20 4.230 ± 0.040 45 21.15 ± 0.204.196 ± 0.039 62 21.53 ± 0.20 4.124 ± 0.038 25 22.49 ± 0.20 3.951 ±0.035 18 22.96 ± 0.20 3.870 ± 0.033 26 23.35 ± 0.20 3.807 ± 0.032 4923.98 ± 0.20 3.707 ± 0.030 29 24.51 ± 0.20 3.629 ± 0.029 52 25.34 ± 0.203.512 ± 0.027 40 25.95 ± 0.20 3.431 ± 0.026 22 26.64 ± 0.20 3.343 ±0.025 14 27.07 ± 0.20 3.291 ± 0.024 15 27.70 ± 0.20 3.218 ± 0.023 1627.88 ± 0.20 3.198 ± 0.022 27 28.30 ± 0.20 3.151 ± 0.022 17 29.04 ± 0.203.072 ± 0.021 12 29.45 ± 0.20 3.030 ± 0.020 14

TABLE 10 Unit Cell Parameters for 4a/L-proline Form D Bravais TypePrimitive Orthorhombic a [Å] 10.092 b [Å] 11.112 c [Å] 30.974 α [deg] 90β [deg] 90 γ [deg] 90 Volume [Å³/cell] 3,473.3 Chiral Contents? ChiralExtinction Symbol P 2₁ 2₁— Space Group(s) P2₁2₁ (18)

Form D was characterized by thermal techniques, proton NMR, HPLC, andDVS. An overlay of the DSC and TGA thermograms for the dried Form D isshown in FIG. 15 . The insignificant weight loss by TGA up to 185° C.and lack of a broad desolvation endotherm by DSC are consistent with ananhydrous/non-solvated material. A small broad endotherm was observed at132° C. (peak maximum). A sharp endotherm with onset at 211° C.corresponds with a steep stepwise weight loss of 26 wt % in the TGAthermogram, likely corresponding with simultaneous melting of theco-crystal and volatilization of L-proline.

Proton NMR data indicated a 1:2 4a/L-proline stoichiometry with 0.2 wt %residual EtOH detected. The purity of 4a in the sample was 99.6% byHPLC.

The DVS isotherm for Form D is shown in FIG. 16 . The material exhibitssignificant hygroscopicity, particularly above 55% RH, with a weightgain of 4.7 wt % noted between 5% and 95% RH. All of this weight waslost on desorption, with minor hysteresis observed. To be noted, thevapor sorption kinetic equilibration timed out on the sorption stepbetween 85%-95% RH, indicating that the co-crystal could potentiallypick up more moisture than what was measured if it was allowed a longerequilibration time.

Example 9: Preparation and Characterization of 4a/L-Proline Form G

Amorphous 4a, pyrazine, and L-proline were combined in 1:20:1 molarratios, respectively, by first dissolving pyrazine in methyl ethylketone and MeOH (90:10, v/v). The pyrazine solution was added to the 4aand L-proline mixture and stirred at room temperature for two days togive an opaque white suspension. Form G was isolated by vacuum filteringthe suspension.

Form G consists of an MEK solvated 4a/L-proline co-crystal. Form Gexhibited a unique crystalline pattern by XRPD (FIG. 17 ) that wasindexed (Table 11).

TABLE 11 Observed peaks for 4a/L-proline Form G °2θ d space (Å)Intensity (%)  8.66 ± 0.20 10.200 ± 0.235  50 10.42 ± 0.20 8.480 ± 0.16247 11.85 ± 0.20 7.461 ± 0.125 43 12.09 ± 0.20 7.316 ± 0.121 27 12.20 ±0.20 7.247 ± 0.118 39 14.62 ± 0.20 6.056 ± 0.082 100 14.93 ± 0.20 5.927± 0.079 45 16.14 ± 0.20 5.485 ± 0.068 7 17.40 ± 0.20 5.092 ± 0.058 8517.85 ± 0.20 4.965 ± 0.055 53 18.22 ± 0.20 4.865 ± 0.053 27 18.31 ± 0.204.842 ± 0.052 25 18.79 ± 0.20 4.719 ± 0.050 64 19.28 ± 0.20 4.600 ±0.047 85 19.43 ± 0.20 4.565 ± 0.047 46 19.81 ± 0.20 4.479 ± 0.045 2220.43 ± 0.20 4.344 ± 0.042 6 20.94 ± 0.20 4.239 ± 0.040 23 21.14 ± 0.204.198 ± 0.039 47 21.59 ± 0.20 4.113 ± 0.038 24 22.15 ± 0.20 4.010 ±0.036 12 22.66 ± 0.20 3.921 ± 0.034 25 23.79 ± 0.20 3.738 ± 0.031 3724.28 ± 0.20 3.663 ± 0.030 34 24.56 ± 0.20 3.622 ± 0.029 23 24.77 ± 0.203.592 ± 0.029 10 25.42 ± 0.20 3.501 ± 0.027 13 25.94 ± 0.20 3.432 ±0.026 15 26.07 ± 0.20 3.415 ± 0.026 15 26.62 ± 0.20 3.345 ± 0.025 1226.80 ± 0.20 3.323 ± 0.024 10 27.33 ± 0.20 3.261 ± 0.023 13 27.89 ± 0.203.197 ± 0.022 7 28.18 ± 0.20 3.165 ± 0.022 10 28.59 ± 0.20 3.120 ± 0.02115 29.08 ± 0.20 3.068 ± 0.021 11 29.54 ± 0.20 3.022 ± 0.020 18

The unit cell volume (Table 12) obtained from indexing the XRPD patternis consistent with a 1:1 4a/L-proline co-crystal with up to 1 mole MEKor pyrazine present (MEK and pyrazine molecules are comparable in volumeand cannot be distinguished by XRPD indexing). The unit cell parameters(Table 12) also indicate that Form G is isostructural to Forms B and C.Form G was confirmed to contain 4a, L-proline, MEK, and minor residualpyrazine in a ˜1:1.2:0.6:0.1 molar ratio by proton NMR.

TABLE 12 Unit Cell Parameters for 4a/L-proline Form G Bravais TypePrimitive Monoclinic a [Å] 10.975 b [Å] 10.310 c [Å] 15.704 α [deg] 90 β[deg] 108.56 γ [deg] 90 Volume [Å³/cell] 1,684.5 Chiral Contents? ChiralExtinction Symbol P 1 2₁ 1 Space Group(s) P2₁ (4)

The single crystal analyses of Forms B and C indicated that those formsare isostructural, with 4a and L-proline forming a channel that housesadditional L-proline for Form B and acetone for Form C. Both theL-proline and the acetone molecules in the respective channels do notform hydrogen bonds with the molecules comprising the channel. Asmentioned above, the space group and other unit cell parameters obtainedfor Form G indicated that it is isostructural to Forms B and C. Althoughother explanations are possible, considering what is known about themolecular packing for those forms and the non-stoichiometric equivalentsof L-proline and MEK measured by proton NMR for Form G, it is highlyprobable that the channel in Form G can accommodate both L-proline andMEK in a non-stoichiometric (and possibly variable) ratio due to theease of exchange created by the lack of hydrogen bonding within thechannel.

Example 10: Purification of 4a by Co-Crystal Formation

A. Purification with L-Proline

Co-crystallization of 4 according to procedures above led to Form B andForm D, and thereby very effectively reduced the amount of f3-anomer 4b.A typical batch of amorphous 4 consisted of 4a/4b in 93.2%/6.3% asdetermined by HPLC. After the co-crystal formation step in a typicalexperiment, the level of 4b was reduced from 6.3% to 2.4% (HPLC). Theresultant 4a/L-proline co-crystal was recrystallized by mixing it withMeOH (2 volumes), and the mixture was heated at reflux for 3 h. Themixture was cooled to 0±3° C. over 2.5 h, and then stirred overnight.The resulting solid was collected by filtration. Afterrecrystallization, the amount of 4b was further reduced to 1.2%, and thepurity of 4a was improved to 98.7% (HPLC).

B. Purification with D-Proline

A separate quantity of amorphous compound 4 contained 89.3% 4a and 10.1%4b as determined by HPLC. Compound 4 (300 mg, 0.670 mmol) and D-proline(77.3 mg, 0.671 mmol) in EtOH (2.4 mL) were heated in a 90° C. oil bath.After refluxing for 15 min, the resulting solution was cooled to roomtemperature in a vial, and kept for 24 h with the vial cap removed tolet EtOH evaporate slowly at room temperature. The precipitated solidwas collected by filtration, and dried in air to give 4a/D-prolineco-crystal. The co-crystal contained 97.6% 4a and 2.1% 4b as determinedby HPLC (150 mg, 40% yield as a white solid). ¹H NMR analysis of theco-crystal indicated a 1:1 molar ratio of 4a and D-proline.

Example 11: Preparation and Characterization of 4a/D-Proline Co-Crystal

A mixture of compound 4a (100 mg, 0.223 mmol) and D-proline (25.8 mg,0.224 mmol) in EtOH (0.8 mL) was heated in a 90° C. oil bath. Afterrefluxing for 15 min, the solution was cooled to room temperature, andkept in a capped vial at room temperature for 24 h. The vial cap wasthen removed to let EtOH evaporate slowly at room temperature. After 24h, the precipitated solid was collected by filtration and then dried inair to give 4a/D-proline co-crystal as a white solid (74 mg, 59% yield)in 99+% purity (HPLC). ¹H NMR analysis of indicated that the co-crystalcontains 1/1 ratio of compound 4a and D-Proline.

The 4a/D-proline co-crystal was characterized by XRPD, DSC, TGA, andDVS. An XRPD pattern for the 4a/D-proline co-crystal was successfullyindexed, indicating the material consists primarily or exclusively of asingle crystalline phase (FIG. 18 , Table 13).

TABLE 13 Observed peaks for 4a/D-proline °2θ d space (Å) Intensity (%) 5.76 ± 0.20 15.323 ± 0.531  14  5.90 ± 0.20 14.964 ± 0.507  13  8.45 ±0.20 10.453 ± 0.247  79  8.74 ± 0.20 10.113 ± 0.231  42  9.22 ± 0.209.579 ± 0.207 57  9.81 ± 0.20 9.007 ± 0.183 20 10.44 ± 0.20 8.470 ±0.162 19 11.55 ± 0.20 7.653 ± 0.132 62 11.77 ± 0.20 7.511 ± 0.127 7512.19 ± 0.20 7.256 ± 0.119 41 12.30 ± 0.20 7.189 ± 0.116 25 13.18 ± 0.206.713 ± 0.101 83 14.04 ± 0.20 6.303 ± 0.089 7 14.52 ± 0.20 6.094 ± 0.08392 14.68 ± 0.20 6.028 ± 0.082 44 15.04 ± 0.20 5.886 ± 0.078 15 15.93 ±0.20 5.559 ± 0.069 26 16.19 ± 0.20 5.470 ± 0.067 40 16.57 ± 0.20 5.345 ±0.064 13 16.95 ± 0.20 5.226 ± 0.061 66 17.27 ± 0.20 5.131 ± 0.059 2817.38 ± 0.20 5.099 ± 0.058 48 17.56 ± 0.20 5.045 ± 0.057 66 17.80 ± 0.204.980 ± 0.056 28 18.22 ± 0.20 4.865 ± 0.053 18 18.43 ± 0.20 4.810 ±0.052 16 18.73 ± 0.20 4.735 ± 0.050 49 19.12 ± 0.20 4.639 ± 0.048 5319.26 ± 0.20 4.604 ± 0.047 36 19.40 ± 0.20 4.573 ± 0.047 40 19.54 ± 0.204.539 ± 0.046 100 19.78 ± 0.20 4.485 ± 0.045 21 20.19 ± 0.20 4.394 ±0.043 33 20.60 ± 0.20 4.308 ± 0.041 8 20.91 ± 0.20 4.245 ± 0.040 3521.23 ± 0.20 4.183 ± 0.039 96 21.41 ± 0.20 4.146 ± 0.038 23 21.62 ± 0.204.107 ± 0.038 32 21.84 ± 0.20 4.066 ± 0.037 11 22.15 ± 0.20 4.009 ±0.036 6 22.37 ± 0.20 3.972 ± 0.035 10 22.59 ± 0.20 3.934 ± 0.034 9 22.73± 0.20 3.910 ± 0.034 20 23.25 ± 0.20 3.823 ± 0.032 26 23.57 ± 0.20 3.771± 0.032 53 23.80 ± 0.20 3.736 ± 0.031 23 23.99 ± 0.20 3.706 ± 0.030 924.23 ± 0.20 3.670 ± 0.030 13 24.32 ± 0.20 3.657 ± 0.030 13 24.58 ± 0.203.619 ± 0.029 28 24.88 ± 0.20 3.575 ± 0.028 14 25.38 ± 0.20 3.506 ±0.027 27 25.76 ± 0.20 3.456 ± 0.026 10 26.11 ± 0.20 3.410 ± 0.026 1526.25 ± 0.20 3.392 ± 0.025 11 26.56 ± 0.20 3.354 ± 0.025 6 26.71 ± 0.203.335 ± 0.025 9 27.01 ± 0.20 3.299 ± 0.024 10 27.41 ± 0.20 3.251 ± 0.02314 27.71 ± 0.20 3.216 ± 0.023 26 27.93 ± 0.20 3.192 ± 0.022 13 28.14 ±0.20 3.168 ± 0.022 17 28.68 ± 0.20 3.110 ± 0.021 9 29.09 ± 0.20 3.067 ±0.021 7 29.30 ± 0.20 3.045 ± 0.020 19

A DSC/TGA overlay for the material is shown in FIG. 19 . The TGAthermogram for the 4a/D-proline co-crystal exhibited two distinct weightloss steps, the first occurring between ˜100° C. and 150-160° C. (7.0%weight loss), and the second between 150 and 230° C. (20.0% weightloss). A broad endotherm was observed by DSC with a peak maximum at 130°C., which coordinates with the first TGA weight loss step, possiblyattributed to the loss of bound solvent/water. Overlapping endothermicevents were observed above ˜170° C., likely due to concurrentmelting/volatilization of the D-proline component of the co-crystal. Asteep drop in the TGA thermogram above ˜250° C. likely corresponds withdecomposition.

A DVS isotherm for the D-proline co-crystal is presented in FIG. 20 .Upon sorption, the co-crystal gained 26 wt % between 5% and 95% RH, withthe vast majority of weight gain occurring between 85% and 95% RH. Thekinetic equilibration timed out during this step, indicating that theco-crystal could potentially pick up more moisture than what wasmeasured if it was allowed a longer equilibration time. Upon desorption,the co-crystal exhibited relatively steady weight loss between 95% and5% RH and lost more weight than was gained during sorption (29 wt %),indicating that the material likely contained solvent/water at the startof the analysis. To be noted, the post-DVS sample of the D-prolineco-crystal was observed to be stuck to the pan and could not berecovered, indicating partial deliquescence during the experiment.

Example 12: Comparative Administration of 4a/L-Proline Material a andAmorphous 4a to Mice

This example evaluated the systemic exposure to 4a after oraladministration of a suspension formulation of Material A as prepared inExample 1 compared to a suspension formulation of amorphous 4a in maleC57BL/6 mice.

Amorphous 4a or Material A was formulated in 0.5% CMC with 5% DMSO(taking into account the presence of proline on a weight basis) andadministered by oral gavage to 35 male C57BL/6 mice at 1000 mg/kg usinga dosing volume of 15 mL/kg. Blood samples for plasma isolation werecollected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-dose, using aseparate group (n=5/group/time point) of mice for each time point, assummarized in Table 14 below. Plasma samples were analyzed forconcentrations of 4a by LC/MS/MS methodology as described below.

TABLE 14 Summary of Mouse Study Dose Dose Blood Dose Level VolumeCollection Group Test Article N Route Vehicle (mg/kg) (mL/kg) Times (hr)1 Amorphous 4a 35 PO 0.5% 1000 15 0.25, 0.5, 1, CMC/5% 2, 4, 8, & 24DMSO in sterile water 2 Material A 35 PO 0.5% 1000 15 0.25, 0.5, 1,CMC/5% 2, 4, 8, & 24 DMSO in sterile water

Reagents and Supplies: All reagents and supplies were of high qualityand of LC/MS grade when appropriate and obtained from standardcommercial suppliers.

Plasma Sample Preparation: 4a was extracted from K₃EDTA-fortified plasmasamples using a protein precipitation method. In a well of apolypropylene microplate (96-well), 20 μL of a 2.5-ng/mL D₃-4a (internalstandard, IS) solution prepared in acetonitrile/H₂O (1:1) was added,followed by addition of 20 μL of plasma sample. The plate was sealedwith sealing tape (Phenomenex, AH0-7362) and gently mixed with avortex-mixer for 1 minute. The solution was pipetted into a well of apolypropylene plate (96-well, 2 mL, Phenomenex, AH0-7194) that contained500 μL of methanol. The plate was sealed and vortex-mixed for 5 minutesfollowed by centrifugation at 3000×g for 3 minutes at room temperature.A 300-4, aliquot of the supernatant was transferred to a new well thatcontained 300 μL of deionized water. After gentle mixing, the plate wassealed and placed in an LC autosampler maintained at 12° C., and aaliquot was injected into an LC/MS/MS system for quantitative analysisof 4a.

Chromatography and Mass Spectrometry Conditions: Liquid chromatographicseparation of 4a was achieved by a reversed-phase analytical column withmobile phase solution containing H₂O, acetonitrile, and formic acid. Thechromatographed analyte was detected by a Waters Xevo TQ-S triplequadrupole mass spectrometer operating in multiple-reaction-monitoring(MRM) mode. Chromatographed peak areas for quality control samples,calibration standards, and study samples were integrated using MassLynxsoftware V4.1 (Waters Corp.).

Pharmacokinetic Analysis: Pharmacokinetic parameter estimates wereobtained from non-compartmental analysis of the mean 4a plasmaconcentration-time data for each dose group using WinNonlin™ softwareversion 6.3 (Pharsight Corp., Cary, NC). The area under the plasmaconcentration-time curve from time zero to the time (t) of the lastmeasurable concentration of 4a (AUC_((0-t))) was determined using thelinear log trapezoidal rule. The time of the last measurableconcentration was defined as the time after which 4a concentrations werebelow the limit of quantitation (BLQ) in the majority of animals foreach dose group.

Results: Mean (±SD) 4a plasma concentration-time data after a singleoral gavage administration of Material A and amorphous 4a are presentedin Table 15 below and displayed in FIG. 21 . Pharmacokinetic parameterestimates for 4a are presented in Table 16.

TABLE 15 Mean (±SD) Plasma Concentrations of 4a in Mice FollowingAdministration of a Single Oral Dose of Material A and Amorphous 4a 4aPlasma Concentration (ng/mL) Time (hr) Material A Amorphous 4a 0.25 9770± 6480 6760 ± 2840 0.5 8090 ± 690  3060 ± 1600 1 733 ± 336 359 ± 140 251.4 ± 27.7 80.7 ± 116  4 15.8 ± 17.9 2.28 ± 0.92 8 3.68 ± 6.12 4.01 ±4.34 24 BLQ^(a) BLQ^(a) ^(a)All samples in group were BLQ.

TABLE 16 Plasma Pharmacokinetic Parameter Estimates after a Single OralAdministration to Mice Dose T_(max) C_(max) AUC_((0-t)) Group (mg/kg)(hr) (ng/mL) (hr*ng/mL) Material A 1000 0.25 9770 5330 Amorphous 4a 10000.25 6760 2880

Example 13: Comparative Administration of 4a/L-Proline Material a andAmorphous 4a to Monkeys

This example evaluated the systemic exposure of Material A as preparedin Example 1 and amorphous 4a formulations, respectively, in malecynomolgus monkeys after a single oral gavage administration at 30 mg/kgusing 0.5% carboxymethylcellulose (CMC) in sterile water as vehicle (5mL/kg) or at 50 mg (regardless of body weight) in a loose-filled capsuleas summarized in Table 17 below.

TABLE 17 Summary of Monkey Study Dose Blood Dose Dose Volume CollectionGroup Test Article N Route Vehicle Level (mL/kg) Times (hr) 1 Material A3 PO 0.5% CMC in 30 mg/kg 5 0.25, 0.5, 1, 2, sterile water 4, 8, 12, and24 2 Amorphous 4a 3 PO 0.5% CMC in 30 mg/kg 5 0.25, 0.5, 1, 2, sterilewater 4, 8, 12, and 24 3 Material A 3 PO Gelatin Capsule 50 mgª NA 0.25,0.5, 1, 2, size 00) 4, 8, 12, and 24 4 Amorphous 4a 3 PO Gelatin Capsule50 mgª NA^(b) 0.25, 0.5, 1, 2, (size 00) 4, 8, 12, and 24 ^(a)Doseadministered regardless of body weight ^(b)NA, Not applicable

Dosing of Material A accounted for the presence of proline on a weightbasis. Blood samples for plasma isolation were collected at 0.25, 0.5,1, 2, 4, 8, 12, and 24 hours post-dose from each monkey. Plasma sampleswere analyzed for concentrations of 4a by LC/MS/MS methodology, andpharmacokinetic analysis was carried out, as described in Example 12above.

Results: Mean (±SD) plasma concentrations after a single oral gavage (30mg/kg, 5 mL/kg) or after capsule dosing (50 mg) are presented in Table18 below and displayed in FIG. 22 . Pharmacokinetic parameter estimatesare presented in Table 19.

TABLE 18 Mean (±SD) 4a Plasma Concentrations in Monkeys Following OralAdministration of Material A or Amorphous 4a as a Suspension or Capsule4a Plasma Concentration (ng/mL) Oral Gavage (30 mg/kg) Capsule (50 mg)Time Material Amorphous Material Amorphous (hr) A 4a A 4a 0.25  264 ±193 103 ± 71 32.1 ± 25.1  15.9 ± 12.9 0.5 265 ± 79 129 ± 92 40.8 ± 16.4 12.3 ± 5.5  1 225 ± 67 128 ± 56 19.6 ± 8.9  5.97 ± 0.95 2 111 ± 39  80.2± 26.4 0.333 ± 0.230^(a) 1.90 ± 1.89 4 11.3 ± 3.3 14.1 ± 5.5 0.349 ±0.259^(a) 0.200^(b) 8  1.60 ± 0.70  1.20 ± 0.45 0.348 ± 0.256^(a) 0.951± 1.30^(a ) 12  0.463 ± 0.228^(c) BLQ^(d) 2.64 ± 3.48^(c)  3.53 ±5.78^(a) 24 BLQ^(e) BLQ^(d) 12.5 ± 11.0^(c)  6.11 ± 9.23^(c) ^(a)2 of 3values for intermediate time point (i.e., between 2 time points withquantifiable values) were below the limit of quantitation (BLQ) andincluded in the mean as ½ the lower limit of quantitation (LLOQ) (i.e.,0.200 ng/mL) ^(b)All values for intermediate time point (i.e., between 2time points with quantifiable values) were BLQ and reported as ½ theLLOQ (i.e., 0.200 ng/mL) ^(c)1 of 3 values was BLQ and assigned a valueof ½ the LLOQ (i.e., 0.200 ng/mL) to calculate mean and standarddeviation ^(d)Mean reported as BLQ because 2 of 3 samples were BLQ^(e)All values were BLQ

TABLE 19 Mean (±SD) Pharmacokinetic Data in Monkeys FollowingAdministration of 4a by Oral Gavage of a Suspension or Capsule T_(max)C_(max) AUC_((0-t)) Group (hr)^(a) (ng/mL) (hr*ng/mL) Material A 0.25 309 ± 117  491 ± 120 (30 mg/kg) Amorphous 4a 0.5 160 ± 78 307 ± 96 (30mg/kg) Material A 0.25 51.0 ± 7.6 110 ± 85 (50 mg capsule) Amorphous 4a0.25 19.7 ± 9.4  81.6 ± 105 (50 mg capsule) ^(a)Data presented as median

Both capsule formulations (but not the oral suspension formulations)produced 4a plasma concentrations that were below or near the lowerlimit of quantitation (i.e., 0.400 ng/mL) at 4 and 8 hours post-dose butdemonstrated a secondary peak of exposure at 24 hours with mean (±SD) 4aconcentrations of 12.5±11.0 and 6.11±9.23 ng/mL for the Material A andamorphous 4a formulations, respectively.

We claim:
 1. A co-crystal ofN-(2-(5-(((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyltetra-hydro-2H-pyran-2-yl)oxy)-3′-fluoro-[1,1′-biphenyl]-2-yl)ethyl)-acetamideand L-proline (1:2), characterized by an X-ray powder diffractogramcomprising the following peaks: 14.76, 16.86, 19.00, and 21.05° 2θ±0.20°2θ as determined on a diffractometer using Cu-Kα radiation at awavelength of 1.54178 Å.